Monospecific and multispecific antibodies and method of use

ABSTRACT

This invention relates to monospecific and multispecific antibodies that may be utilized for the diagnosis and treatment of various diseases. In addition, these antibodies may be modified by protease cleavage. Protease control or regulation may be provided by a protease site located in, for example, a linker. These protease-regulated antibodies may also be utilized for the diagnosis and treatment of various diseases.

This application claims benefit to U.S. Provisional Application Ser. No. 60/955,912, filed Aug. 15, 2007, and U.S. Provisional Application Ser. No. 60/955,913, filed Aug. 15, 2007, the contents of which are incorporated herein by reference in their entirety.

FIELD OF THE INVENTION

This invention relates to monospecific and multispecific antibodies that may be utilized for the diagnosis and treatment of various diseases. In addition, these antibodies may be modified by protease cleavage. Protease control or regulation may be provided by a protease site located in, for example, a linker. These protease-regulated antibodies may also be utilized for the diagnosis and treatment of various diseases.

BACKGROUND OF THE INVENTION

An antibody may be directed against one or more different antigens or one or more different epitopes on the same antigen. For example, a bispecific antibody is directed against two different antigens or two different epitopes on the same antigen. As bispecific antibodies can simultaneously bind to two distinct targets, these antibodies have great potential for antibody-based diagnosis and for the treatment of various diseases and disorders such as cancer, infectious diseases, autoimmune diseases, and blood diseases. For example, bispecific antibodies can selectively stimulate and expand T lymphocytes (Wong, et al., J. Immunol. 139:1369-1374, 1987; Wong, et al., Clin. Immunol. Immunopathol. 58:236-250, 1991), direct immune cells or toxic agents to kill tumor cells (Lum, et al., Exp. Hematol. 34:1-6,2006; Wolf, et al., Drug Discov. Today 10:1237-1244, 2005; Cao, et al., Adv. Drug Deliv. Rev. 55:171-197, 2003; Talac, et al., J. Biol. Regul. Homeost. Agents 14:175-181, 2000), and simultaneously block two receptors (Lu, et al., J. Biol. Chem. 279:2856-2565, 2004). In addition, a bispecific antibody may be used as a substitute for Factor VIII to enhance enzymatic reaction (US Patent Application No. 2007/0041978) or to direct stem cells to the site of injury in patients with myocardial infarction (Lum, et al., Blood Cells Mol. Dis. 32:82-87, 2004).

Bispecific antibodies targeting tumor-associated antigens and toxic agents may be used in cancer therapy. For example, using this technology, one arm of the bispecific antibody may be directed to a tumor-associated antigen such as Her2, EGF receptor, CD20, CD22, CD30, CD33, CD52, and CA-125, and the other arm of the bispecific antibody may target a toxin, drug, or cytokine. That is, bispecific antibodies may selectively direct toxic agents to tumor cells enhancing the efficacy of therapeutic antibodies and decreasing systemic toxicity. Examples of toxin/drug include calicheamicin, doxorubicin, epirubicin, methotrexate, ricin A, saporin, gelonin, and vinca alkaloids, and cytokine examples include tumor necrosis factor alpha (TNF-alpha) and IL-2.

Specific cleavage by proteases of defined sites in biologically important effector proteins is a well known method for the natural control of cellular and extracellular physiological processes. Examples include protease activation and inhibition of the coagulation cascade (Butenas, et al., Biochemistry 67:3-12, 2002; Esmon, Chest, 124:26S-32S, 2003), protease activation of protease-activatable receptors (Coughlin, Arterioscler. Thromb. Vasc. Biol. 18:514-518, 1998), protease release of membrane associated cytokines (Amour, et al., FEBS Lett. 435:39-44, 1998), protease processing of prohormones in secretory vesicles (Moore, et al., Arch. Physiol. Biochem. 110:16-25, 2002), and protease processing of proproteins during secretion (Scamuffa, et al., FASEB J. 20:1954-1963, 2006). Proteases are often expressed or located in a tissue-specific or tumor-specific manner and examples include the membrane serine protease corin in heart tissue (Yan, et al., Proc. Natl. Acad. Sci. USA 97:8525-8529, 2000), the kallikrein serine protease prostate-specific antigen (PSA) in prostate tissue, prostate cancer, and seminal fluid (Veveris-Lowe, et al., Semin. Thromb. Hemost. 33:87-99, 2007), the membrane serine protease hepsin in liver tissue and tumors (Xuan, et al., Cancer Res. 66:3611-3619, 2006), coagulation protease factor X expressed in the liver and secreted into blood (Miao, et al., J. Biol. Chem. 267:7395-7401, 1992), and digestive proteases expressed in the pancreas and released to the duodenum (Belorgey, et al., Biochem. J. 313:555-560, 1996). Specific cleavage of amino acid sequences by human proteases include thrombin (Chang, Eur. J. Biochem. 151:217-224,1985), factor Xa (Nagai, et al., Methods Enzymol. 153:461-481, 1987), furin (Brennan, et al., FEBS Lett. 347:80-84, 1994), subtilisin-like prohormone convertases (Lipkind, et al., J. Biol. Chem. 270:13277-13284, 1995), and the matrix metalloproteinases (Minod, et al., J. Biol. Chem. 281:38302-38313, 2006). Genes encoding specific proteases may be up-regulated in tumor tissue and Table 2 indicates proteases that are associated with cancer tissue.

Protease cleavage is widely used in in vitro studies to specifically remove protein or peptide tags from recombinant proteins or to process hybrid recombinant proteins. For example, human rhinovirus 3C protease, thrombin, or factor Xa have been used to remove glutathione S-transferase (GST) tags (Dian, et al., Life Sciences News—Amersham Biosciences 10:1-5, 2002) and factor Xa has been use to process hybrid proteins (Nagai, et al., 1987). Proteases are often targets for drugs as a means to regulate biological processes; and examples include factor Xa (Phillips, et al., J. Med. Chem. 41:3557-3562,1998), thrombin (Riester, et al., Proc. Natl. Acad. Sci. USA 102:8597-8602, 2005), urokinase (Killeen, et al., Br. J. Cancer 96:262-268, 2007), and factor VIIa (Kohrt, et al., Bioorg. Med. Chem. Lett. 15:4752-4756, 2005). Finally, proteins developed as biological drugs may be modified to prevent cleavage by proteases and to improve their stability in vitro or in vivo (Light, et al., Eur. J. Biochem. 262:522-533, 1999; Saenko, et al., Haemophilia 12:42-51, 2006).

Specific protease cleavage sites have been incorporated into linkers that link a toxin molecule to a targeting antibody in order to allow protease specific release of the toxin by intracellular proteases (Trail, et al., Cancer Immunol. Immunother. 52:328-337, 2003). Furthermore, targeting antibodies have been created in many formats. For example, bispecific antibodies have been developed to allow binding to two different antigens or two different epitopes of an antigen by a single antibody molecule (Segal, et al., Curr. Opin. Immunol. 11:558-562, 1999; Tomlinson, et al., Methods Enzymol. 326:461-479, 2000; Wu, et al., Nat Biotechnol. 25:1290-1297, 2007). Other bispecific molecules have been generated with the ability to block two receptors (Lu, et al., J. Biol. Chem. 279:2856-2865, 2004) and to recruit immune cells to attack cancer cells and tumor tissue (Loffler, et al., Leukemia 17:900-909, 2003; Lum, et al., Exp. Hematol. 34:1-6, 2006).

The present invention relates to a novel antibody format, for example, monospecifc and multispecific antibodies. The antibodies of the present invention may be constructed by tandem linking of two different heavy chain (H) variable region domains (V_(H)) and two different light chain (L) variable region domains (V_(L)). The heavy chain and light chain may form a Fab-like or IgG-like molecule through the disulfide bond between constant (C) regions. Multispecific antibodies may be generated by linking more than two antibody variable domains.

The antibodies of the present invention may be modified by protease cleavage. These protease-regulated antibodies may be, for example, monospecific antibodies, bispecific antibodies, or antibodies with sequential binding-activity upon protease digestion in either, for example, Fab-like or IgG-like format. Protease control or regulation may be provided by a protease site located in, for example, a linker. These protease-regulated antibodies may be utilized for the diagnosis and treatment of various diseases, and provide an additional level of control for biological drugs for therapeutic or diagnostic applications.

DESCRIPTION OF THE FIGURES

FIG. 1. Schematic drawing of a monospecific protease-regulated antibody with a linker which contains a protease site between variable domain and Fc domain (“Type 1”).

FIG. 2. Schematic drawing of a bispecific protease-regulated antibody with a linker which contains a protease cleavage sequence that allows removal of one antigen-binding site (“Type 2”).

FIG. 3. Schematic drawing of another bispecific protease-regulated antibody with a linker which contains a protease cleavage sequence that allows removal of one antigen-binding site (“Type 2”).

FIG. 4. Schematic drawing of the application of a bispecific protease-regulated antibody that simultaneously binds two different antigens.

FIG. 5. Schematic drawing of a protease-regulated antibody that cannot bind to two different antigens simultaneously (“Type 3”).

FIG. 6. Schematic drawing of the application of a protease-regulated antibody that cannot bind to two different antigens simultaneously.

FIG. 7. Schematic drawing of a monospecific protease-regulated antibody ‘prodrug’ that can only bind antigen following protease activation to remove inactive blocking antibody domains (“Type 4”).

FIG. 8. Map of an expression vector for an IgG-like bispecific antibody. SignalP: signal peptide; VLam3E10: variable region of 3E10 lambda chain; Vk19G9: variable region of 19G9 kappa chain; C kappa: constant region of kappa chain; DHFR: dihydrofolate reductase; V_(H): variable region of heavy chain; Neo: neomycin resistant gene; 3E10VH: variable region of 3E10 heavy chain; 19G9VH: variable region of 19G9 heavy chain; CH: constant region of heavy chain; Amp: ampicillin resistant gene.

FIG. 9. Map of expression vector of Fab-like bispecific antibody. LacZ, lac Z promoter; ompA and pho A, signal peptide; VL-link1-VK, variable region of light chain of bispecific antibody against tissue factor and RG1; CL-kappa, constant region of kappa chain; VHs with linker, variable region of heavy chain of bispecific antibody against tissue factor and RG1; CH1, the first constant region of IgG heavy chain; cat, chloramphenicol resistant gene.

FIG. 10. TF-binding ELISA. Four bispecific antibodies and parental antibodies were analyzed for binding to TF. Antibodies were detected with HRP-conjugated anti-human IgG was used for detection. Curve fitting of the data was performed using a 4-parameter equation with the Solver function in Microsoft Excel. Positive control anti-TF IgG 3E10x: IC₅₀=2.0 nM (filled diamond, solid line); Linker 1 (SEQ ID NO: 1) IgG-like bispecific antibody: IC₅₀=0.78 nM (filled triangle, large dashed line); Linker 2 (SEQ ID NO: 2) IgG-like bispecific antibody: IC₅₀=0.93 nM (open triangle, small dashed line); Linker 3 (SEQ ID NO: 3) IgG-like bispecific antibody: IC₅₀=1.06 nM (filled square, alternating small and large dashed line); Linker 4 (SEQ ID NO: 4) IgG-like bispecific antibody: IC₅₀=1.01 nM (open square, two large and one small dashed line); and negative control anti-RG1 IgG 19G9: no binding (open diamond, solid line).

FIG. 11. RG1-binding ELISA. A bispecific antibody containing Linker 1, anti-RG1 antibody 19G9, and polyclonal nonimmune control human IgG kappa were analyzed for binding to RG-1. Curve fitting of the data was performed using a 4-parameter equation with the Solver function in Microsoft Excel. Positive control anti-RGI IgG 19G9: IC₅₀=1.4 nM (filled diamond, solid line); Linker 1 (SEQ ID NO: 1) IgG-like bispecific antibody: IC₅₀=1.1 nM (filled triangle, large dashed line); and negative control nonimmune polyclonal human IgG kappa: no binding (open triangle, small dashed line).

FIG. 12. Measurement of the antigen-binding activity of a bispecific protease-regulated antibody using a sandwich antigen-binding ELISA. The linker of this antibody contained cleavage sites for enterokinase.

FIG. 13. Measurement of the antigen-binding activity of protease-regulated antibodies 3E10-Type1-Fab and 19G9-Typel -Fab. The controls are designated 3E10-Reg-Fab, 19G9-Reg-Fab, and HuFab.

FIG. 14. Measurement of the antigen-binding activity of Fab-like protease-regulated antibodies H1L1, H1L4, H1L7, H4L7, and H5L5 (Type 2) in the absence and presence of enterokinase. Parental antibodies 3E10 and 19G9, and polyclonal human Fab were used as control.

FIG. 15. Measurement of the antigen-binding activity of Fab-like protease-regulated antibodies H2L1, H2L2, and H2L8 (Type 3) and H3L1, H3L4, and H5L4 (Type 4) in the absence and presence of enterokinase. Parental antibodies 3E10 and 19G9, and polyclonal human Fab were used as control.

FIG. 16. Western blots of protease-regulated antibody 3E10-Type1-Fab detected with anti-Myc antibody (A) or anti-kappa chain antibody (B). Lane 1 and 2: 3E10-Type1-Fab without or with enterokinase digestion, respectively. Lane 3 and 4: 3E10-Reg-Fab without or with enterokinase digestion, respectively.

FIG. 17. Western blots of Fab-like protease-regulated antibodies H1L1, H1L7, and H5L5 (Type 2) in the absence and presence of enterokinase. Antibodies were detected with anti-IgG(H+L) antibody. Lane I and 2: H1L1 without or with enterokinase digestion, respectively. Lane 3 and 4: H1L7 without or with enterokinase digestion, respectively. Lane 5 and 6: H5L5 without or with enterokinase digestion, respectively. Lane 7: 3E10-Reg-Fab.

FIG. 18. Western blots of Fab-like protease-regulated antibodies H2L2 and H2L8 (Type 3) and H3L4 (Type 4) in the absence and presence of enterokinase. Antibodies were detected with anti-Myc antibody. Lane 1 and 2: H2L2 without or with enterokinase digestion, respectively. Lane 3 and 4: H2L8 without or with enterokinase digestion, respectively. Lane 5 and 6: H3L4 without or with enterokinase digestion, respectively.

DESCRIPTION OF THE INVENTION

It is to be understood that this invention is not limited to the particular methodology, protocols, cell lines, animal species or genera, constructs, and reagents described and as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims.

It must be noted that as used herein and in the appended claims, the singular forms “a,” “and,” and “the” include plural reference unless the context clearly dictates otherwise. Thus, for example, reference to “an antibody” is a reference to one or more antibodies and includes equivalents thereof known to those skilled in the art, and so forth.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this invention belongs. Although any methods, devices, and materials similar or equivalent to those described herein can be used in the practice or testing of the invention, the preferred methods, devices and materials are now described.

All publications and patents mentioned herein are hereby incorporated herein by reference for the purpose of describing and disclosing, for example, the constructs and methodologies that are described in the publications which might be used in connection with the presently described invention. The publications discussed above and throughout the text are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention.

For convenience, the meaning of certain terms and phrases employed in the specification, examples, and appended claims are provided below.

“Antibody” as used herein includes intact immunoglobulin molecules (e.g., IgG1, IgG2a, IgG2b, IgG3, IgM, IgD, IgE, IgA), as well as fragments thereof, such as Fab, F(ab′)₂, scFv, Fv, and diabody which are capable of specific binding to an epitope of a protein. The term antibody also extends to other protein scaffolds that are able to orient antibody complementarity-determining region (CDR) inserts into the same active binding conformation as that found in natural antibodies such that the binding to the target antigen observed with these chimeric proteins is maintained relative to the binding activity of the natural antibody from which the CDRs were derived.

“Antibody fragments” comprise a portion of a full length antibody, generally the antigen binding or variable domain thereof. Examples of antibody fragments include Fab, Fab′, F(ab′)₂, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; monospecific antibodies; bispecific antibodies; and multispecific antibodies formed from antibody fragments.

The term “autoimmune diseases” includes, but is not limited to, multiple sclerosis, rheumatoid arthritis, lupus, type I diabetes mellitus, Crohn's disease, autoimmune hemolytic anemia, autoimmune hepatitis, glomerulonephritis, inflammatory bowel disease, myocarditis, psoriasis, thyroiditis, ulcerative colitis, and Graves'disease.

The terms “biological sample” or “patient sample” as used herein, refers to a sample obtained from an organism or from components (e.g., cells) of an organism. The sample may be of any biological tissue or fluid. The sample may be a “clinical sample” which is a sample derived from a patient. Such samples include, but are not limited to, sputum, blood, serum, plasma, blood cells (e.g., white cells), tissue samples, biopsy samples, urine, peritoneal fluid, and pleural fluid, saliva, semen, breast exudate, cerebrospinal fluid, tears, mucous, lymph, cytosols, ascites, amniotic fluid, bladder washes, and bronchioalveolar lavages or cells therefrom, among other body fluid samples. The patient samples may be fresh or frozen, and may be treated with heparin, citrate, or EDTA. Biological samples may also include sections of tissues such as frozen sections taken for histological purposes.

The term “cancer” includes, but is not limited to, solid tumors, such as cancers of the breast, respiratory tract, brain, reproductive organs, digestive tract, urinary tract, eye, liver, skin, head and neck, thyroid, parathyroid, and their distant metastases. The term also includes lymphomas, sarcomas, and leukemias.

The term “ conjugate” refers to an antibody chemically linked to a chemical moiety, such as a therapeutic or cytotoxic agent.

The term “infectious diseases” includes, but is not limited to, HTV/AIDS, lower respiratory infections, diarrheal diseases, tuberculosis, malaria, measles, pertussis, tetanus, meningitis, syphilis, hepatitis B and tropical diseases.

The term “linker” refers to a peptide (or polypeptide) comprising two or more amino acid residues joined by peptide bonds and used to link one or more antibody domains. The linker may contain one or more protease cleavage sites.

The term “protease” refers to any enzyme, including the endopeptidases and exopeptidases, which catalyze the hydrolytic breakdown of proteins into peptides or amino acids.

The present invention is directed to the design and production of monospecific, bispecific antibodies, and multispecific antibodies. For example, the bispecific antibodies and multispecific antibodies may comprise tandem linked V_(H)a-V_(H)b-V_(H)c . . . C_(H) in one polypeptide and V_(L)a-V_(L)b-V_(L)c . . . C_(L) in another polypeptide. Alternately, the V_(H) and V_(L) domains may be exchanged from one polypeptide to another to create polypeptides such as V_(H)a-V_(L)b-V_(H)c . . . C_(H) and V_(L)a-V_(H)b-V_(L)c . . . C_(L). The two polypeptides may form □immers in the Fab format or the half IgG-like format, or two of each polypeptide may form a four polypeptide-containing homodimer of the IgG-like format. These bispecific or multispecific antibodies or antibody fragments thereof may simultaneously bind different antigens or different epitopes of the same antigen.

As an example, a recombinant IgG-like bispecific antibody may be constructed by the tandem linking of two different V_(H) domains of a heavy chain and two different V_(L) domains of a light chain. The construct is exemplified as follows:

-   -   heavy chain=NH₂-V_(H)1-V_(H)2-C_(H)1-C_(H)2-C_(H)3-COOH     -   light chain=NH₂-V_(L)1-V_(L)2-C_(L).

Another bispecific antibody may comprise the following:

-   -   heavy chain=NH₂-V_(L)1-V_(H)2-C_(H)1-C_(H)2-C_(H)3-COOH     -   light chain=NH₂-V_(H)1-V_(L)2-C_(L)-COOH.

The present invention also relates to protease-regulated antibodies. Protease-regulated antibodies may be, for example, monospecific antibodies, bispecific antibodies, multispecific antibodies, or antibodies with sequential binding-activity upon protease digestion in either, for example, Fab-like or IgG-like format. Protease control or regulation may be provided by a selective protease site located in, for example, a linker. These protease-regulated antibodies may be utilized for the diagnosis and treatment of various diseases including but not limited to cancer, infectious disease, and autoimmune diseases, and provide an additional level of control for biological drugs for therapeutic or diagnostic applications.

Protease-regulated antibodies may comprise a heavy chain (H) variable domain (V_(H))-linker-heavy chain constant domain (CH) in one polypeptide and a light chain (L) variable domain (V_(L))-linker-light chain constant domain (CL) in another polypeptide. Bispecific protease-regulated antibodies may comprise, for example, V_(H1)-linker-V_(H2)-CH in one polypeptide and V_(L1)-linker-V_(L2)-CL in another polypeptide, both regulated by proteolytic cleavage of the linker. Alternately, the V_(H) and V_(L) domains in the bispecific protease-regulated antibodies may be exchanged from one polypeptide to another polypeptide to create polypeptides such as, for example, V_(H1)-linker-V_(L2)-CH and V_(L1)-linker-V_(H2)-CL. The two polypeptides may form □immers, for example, in a Fab-like format, a half-IgG-like format, or an IgG-like format (e.g., two of each polypeptide forming a four polypeptide-containing homodimer). The bispecific and sequential protease-regulated antibodies or antibody fragments may (1) simultaneously bind two different antigens or different epitopes of the same antigen, (2) sequentially bind two different antigens or different epitopes on the same antigen in a manner that may be dependent on the length, adjacent sequence, and design of the linker, or (3) a monospecific protease-activated binder which is in latent or prodrug form prior to protease digestion and which is switched on by protease cleavage. Libraries of bispecific protease-regulated antibodies in the Fab-like format can be readily created, expressed in bacteria, and screened for specific functionalities, including susceptibility of the linker to cleavage by a specific protease and optimization of this cleavage step.

Several types of protease-regulated antibodies are described herein whereby antibody formats are designed with selectivity due to specific protease-dependent binding or protease-specific functionality. In particular, these protease-regulated antibodies may be described by deletions and/or additions of antibody framework, location of the linker, and its properties including length and solvent accessibility. Furthermore, the linker may contain a cleavage site specific for a protease found in a target cell or tissue. One example of a protease-regulated antibody may contain a protease site in a linker located between the variable domain and the constant region domain and this antibody may bind only one antigen as illustrated in FIG. 1 (“Type 1”).

Another example of a protease-regulated antibody may simultaneously bind two different antigens or two different epitopes as shown in FIGS. 2 and 3 (“Type 2”) in the absence of a protease. The first V_(H)/V_(L) domains of this antibody bind to an antigen without blocking the second V_(H)/V_(L) domains from binding to a second antigen. This antibody can bind to antigens without steric blocking of the CDR regions of the second variable domains. The simultaneous binding of this protease-regulated antibody to both antigens is prevented by proteolytic cleavage. That is, when the linker is cleaved by a protease, the antibody can only bind to the second antigen or separately bind two antigens. Simultaneous antigen binding is important for antibody function, for example, in cross-linking receptors, which may be prevented by proteolytic cleavage. Thus, an additional degree of specificity is added by including a protease site in the linker.

This bispecific protease-regulated antibody is more selective than a monospecific antibody because this antibody will specifically target cells or tissues expressing both antigens. The additional degree of specificity is provided by the specific protease site in the linker. In FIG. 4, Cell A and Cell B express both Antigen 1 and Antigen 2, however only Cell B expresses the selective protease. The bispecific protease-regulated antibody with the uncleavable linker (i.e., this linker does not contain the protease cleavage site) will bind to Cell A with greater avidity because the bispecific antibody is able to bind to both antigens. In contrast, the bispecific protease-regulated antibody will bind to Cell B with lower avidity because the Antigen 1 binding domain is removed by proteolytic cleavage of the linker by the selective protease expressed by Cell B (alternately, the selective protease may be expressed by adjacent cells localized in the same tissue as Cell B).

In contrast, FIG. 5 illustrates a protease-regulated antibody that may sequentially bind to each antigen in a protease-dependent manner. That is, prior to protease cleavage of the linker, the protease-regulated antibody binds to a first antigen and following protease cleavage, the antibody binds to a second antigen (“Type 3”). The V_(H)/V_(L) domains of the N-terminal antibody bind to an antigen, but block the CDR regions of the downstream V_(H)/V_(L) domains from binding to a second antigen. Protease cleavage of the linker allows removal of the N-terminal antibody, and removing the N-terminal antibody domains then permits binding to a second antigen. This allows for greater cell and/or tissue selectivity by requiring sequential binding.

In FIG. 6, Cell A and Cell B express both Antigen 1 and Antigen 2, but only Cell B expresses the selective protease. In addition, Antigen 2 is a cell surface receptor that internalizes into the cell and allows internalization of antibodies that bind to it. The protease-regulated antibody will bind to Antigen 1 expressed by Cell A and Cell B. However, only Cell B expresses the selective protease (or possibly cells adjacent to Cell B in the same tissue). The protease-regulated antibody will be activated by proteolytic cleavage and internalized via Antigen 2 expressed on Cell B. Thus, this protease-regulated antibody will be specifically internalized by cells expressing Antigen 1, Antigen 2, and the selective protease.

In an additional example, a protease-regulated antibody may not bind to an antigen before protease digestion, but may bind to antigen following protease digestion (“Type 4”). An example of this antibody is illustrated in FIG. 7. This monospecific protease-regulated antibody also contains a protease cleavage linker that allows removal of the N-terminal non-functional antibody which then leads to binding to an antigen by the functional antibody domains that are thus exposed. Type 4 protease-regulated antibodies may be created by three approaches. In the first approach the protease cleavable linker sequence is modified so that it prevents the N-terminal V_(H) and V_(L) domains of a Type III antibody (V_(H)1 and V_(L)1) from binding to the first antigen. Examples of these linkers are shown in the sequences in Table 8. In the second approach, the linkers utilized in Type III antibodies shown in Tables 6 and 7 are now combined with heterodimeric N-terminal V_(H) and V_(L) domains that have been mutated to destroy their antigen binding function. Examples of this approach are shown in the sequences in Table 9 in which the CDR3 of V_(H)1 and CDR3 of V_(L)1 are replaced by a poly-alanine sequence of a similar length as the respective CDR. In the third approach, the linkers utilized in Type III antibodies shown in Tables 6 and 7 are combined with homodimeric N-terminal domains derived from the constant regions of antibodies. For example, the complete V_(H)1 and V_(L)1 domains of a Type III antibody are both replaced by the same constant domain that is capable of heterodimerization, for example, the CH3 domain of IgG or the CH4 domain of IgE.

These protease-regulated antibodies may be modified by protease cleavage of the linker as described below. For example, the protease-regulated antibody illustrated in FIG. 1 (Type 1) contains a protease site in the linker between the antigen binding domains and the Fc domain. This antibody will specifically target cells or tissues that present the antigen. When the linker is cleaved by the protease, the resultant protease-regulated antibody releases the functional Fc portion. In tissues where the protease is present, this antibody will release the Fc portion which is essential to antigen crosslinking, and induce an immune response such as antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). To illustrate, hepsin, a serine protease, is expressed in both tumor tissue and normal liver tissue. In a cancer patient treated with a protease-regulated antibody against hepsin, the antibody would attack the tumor cells via the Fc portion-induced ADCC and CDC. However, in the liver, the protease-regulated antibody would initially bind to hepsin, but the Fc portion would be cleaved by a liver-specific protease prior to initiation of ADCC or CDC preventing liver toxicity.

The peptide (or polypeptide) linker of the protease-regulated antibody may comprise two or more amino acid residues and may contain one or more protease cleavage sites. The linkers may alter antibody conformation, stability, and antigen-binding activities. The length of linkers may range, for example, from 0 to about 100 amino acid residues. The following are examples of linkers:

Linker 1: SDDDDK (SEQ ID NO: 1) Linker 2: GGGGSDDDDK (SEQ ID NO: 2) Linker 3: GGGGSDDDDKGGGGS (SEQ ID NO: 3) Linker 4: GGGGSGGGGSGGGGS (SEQ ID NO: 4) Linker 5: IHPVLSGLSRIVNGEDAVPG (SEQ ID NO: 5) Linker 6: VAAPFDDDDKIVGGYICEEN (SEQ ID NO: 6) Linker 7: ELLESYIDGRIVEGSDAEIG (SEQ ID NO: 7) Linker 8: STQSFNDFTRVVGGEDAKPG (SEQ ID NO: 8) Linker 9: PERGDNNLTRIVGGQECKDG (SEQ ID NO: 9) Linker 10: EDQEDQVDPRLIDGKMTRRG (SEQ ID NO: 10) Linker 11: KRNASKPQGRIVGGKVCPKG (SEQ ID NO: 11) Linker 12: SVCTTKTSTRIVGGTNSSWG (SEQ ID NO: 12) Linker 13: SRIVG (SEQ ID NO: 13) Linker 14: GSLVSGSCSQIINGEDCSPH (SEQ ID NO: 14) Linker 15: SRIIN (SEQ ID NO: 15) Linker 16: NKLVH (SEQ ID NO: 16) Linker 17: DKIID (SEQ ID NO: 17) Linker 18: FNVLG (SEQ ID NO: 18) Linker 19: TRAIG (SEQ ID NO: 19) Linker 20: TRLDP (SEQ ID NO: 20) Linker 21: TRIIK (SEQ ID NO: 21) Linker 22: SGSNQ (SEQ ID NO: 22) Linker 23: SKVLN (SEQ ID NO: 23) Linker 24: NKIIG (SEQ ID NO: 24) Linker 25: DKLLE (SEQ ID NO: 25)

Table 1 illustrates the excision site of several proteases.

TABLE 1 Cleavage Enzyme/ Excision site ↓ Self-Cleavage Asp-Asp-Asp-Asp-Lys↓ Enterokinase (DDDDK) (SEQ ID NO: 26) Ile-Glu/Asp-Gly-Arg↓ Factor Xa  (IE/DGR) protease (SEQ ID NO: 27) Leu-Val-Pro-Arg↓Gly-Ser Thrombin (LVPR | GS) (SEQ ID NO: 28) Glu-Asn-Leu-Tyr-Phe-Gln↓Gly TEV protease (ENLYFQ | G) (SEQ ID NO: 29) Leu-Glu-Val-Leu-Phe-Gln↓Gly-Pro Human rhinovirus (LEVLFQ | GP) 3C protease (SEQ ID NO: 30) Ser-Ser-Val-Phe-Ala-Gln↓Ser-Ile- PCSK9 (NARC-1) Pro (SSVFAQ | SIP) (SEQ ID NO: 31) Lys-Gln-Leu-Arg↓Val-Val-Asn-Gly Hepsin (KQLR | VVNG) (SEQ ID NO: 32) Specific intein-encoded Intein 1 & sequences intein 2 Signal sequences Signal peptidases

The cleavage sites of additional proteases that may be incorporated in a linker are described in Table 2.

TABLE 2 TUMOR ASSOCIATED PROTEASES (Extracellular Or Intracellular) ADAM metallopeptidase domain 9 (meltrin gamma) ADAM metallopeptidase domain 10 ADAM metallopeptidase domain 17 (TNFalpha, converting enzyme) ADAM metallopeptidase domain 28 ADAM-like, decysin 1 ADAM metallopeptidase, thrombospondin type 1 motif 1 ADAM metallopeptidase, thrombospondin type 1 motif 5, aggrecanase-2 ADAMTS-like 3 ADAMTS-like 4 Beta-site APP-cleaving enzyme 1 Bleomycin hydrolase Bone morphogenetic protein 1 Complement component 1, r subcomponent Complement component 1, s subcomponent Calpain 2, (m/II) large subunit Caspase 1, apoptosis-related cysteine peptidase (IL-1β convertase) Caspase 2, apoptosis-related cysteine peptidase Caspase 3, apoptosis-related cysteine peptidase Caspase 4, apoptosis-related cysteine peptidase Caspase 6, apoptosis-related cysteine peptidase Caspase 7, apoptosis-related cysteine peptidase Caspase 9, apoptosis-related cysteine peptidase Complement factor D (adipsin) CASP8 and FADD-like apoptosis regulator Cathepsin B Cathepsin F Cathepsin H Cathepsin K Cathepsin L Cathepsin L2 Cathepsin O Cathepsin S Cylindromatosis (turban tumor syndrome) Extra spindle pole bodies homolog 1 (S. Cerevisiae) Granzyme A (granzyme 1, CTL-associated serine esterase 3) Histocompatibility (minor) 13 Hepsin (transmembrane protease, serine 1) HtrA serine peptidase 1 Kallikrein-related peptidase 11 Legumain Lon peptidase 1, mitochondrial Mucosa associated lymphoid tissue lymphoma translocation gene 1 Membrane-bound transcription factor peptidase, site 1 Matrix metallopeptidase 1 (interstitial collagenase) Matrix metallopeptidase 12 (macrophage elastase) Matrix metallopeptidase 14 (membrane-inserted) Matrix metallopeptidase 9 (gelatinase B, 92 kDa type IV collagenase) N-acetylated alpha-linked acidic dipeptidase-like 1 Napsin A aspartic peptidase Pregnancy-associated plasma protein A, pappalysin 1 Proprotein convertase subtilisin/kexin type 5 Plasminogen activator, tissue Plasminogen activator, urokinase Peptidase (mitochondrial processing) beta Protease, serine, 3 (mesotrypsin) Protease, serine, 8 (prostasin) Proteasome (prosome, macropain) subunit, alpha type, 1 Proteasome (prosome, macropain) subunit, alpha type, 6 Proteasome (prosome, macropain) subunit, beta type, 4 Proteasome (prosome, macropain) subunit, beta type, 9 Proteasome (prosome, macropain) subunit, beta type, 10 SUMO1/sentrin specific peptidase 1 Suppression of tumorigenicity 14 (colon carcinoma) Tubulointerstitial nephritis antigen Torsin family 1, member A (torsin A) Tripeptidyl peptidase I Tripeptidyl peptidase II Tryptase alpha/beta 1 Tryptase alpha/beta 1 Ubiquitin specific peptidase 4 (proto-oncogene) Ubiquitin specific peptidase 10 Ubiquitin specific peptidase 11 Ubiquitin specific peptidase 14 (tRNA-guanine transglycosylase) Ubiquitin specific peptidase 15 Ubiquitin specific peptidase 16 Ubiquitin specific peptidase 18 Ubiquitin specific peptidase 25 YME1-like 1 (S. cerevisiae) Zinc metallopeptidase (STE24 homolog, yeast)

The protease-regulated antibodies of the present invention may bind one or more antigens. These antigens may be selected from the group consisting of cytokines, cell surface receptors, enzymes, and receptors. These antigens include, but are not limited to, CD3, CD4, CD8, CD20, CD25, CD28, CD33, CD52, IL-2, IL-7, IL-8, TNF-alpha, TGF-beta INF-beta, INF-gamma, GMCSF, GCSF, VEGF, C5, EpCAM, EGF receptor, CD2 receptor, IL 2 receptor, IgE receptor, intergrin, and MHC class II.

The antibodies of the present invention may be utilized for the diagnosis and therapy of various diseases. For example, antibodies directed against human immunological cells and tumor-associated antigen may be used for cancer therapy. These antibodies may also be directed against tumor-associated antigen and toxic agents or enzymes for use as a cancer therapeutic. The antibodies of the present invention may also be utilized for the treatment of hemophilia and thrombosis as well as stem cell transplantation. These antibodies may be used for the selective stimulation and expansion of lymphocyte subset. In addition, these antibodies may used for the detection of disease-related antigens.

For cancer immunotherapy, bispecific antibodies may be used to recruit the immune system to attach tumor cells. Targets on immunological cells include, but are not limited to, CD3, CD8, and Fc receptor. Tumor-associated antigens include, but are not limited to, Her2, EGF receptor, CD20, CA-125, and carcinoembryonic antigen (CEA). For example, a bispecific antibody against CD8 and Her2 can direct CD8-expressing cytotoxic lymphocytes to attack Her2 expressing breast cancer cells.

The antibodies or antibody fragments of the invention, or compositions including the antibodies or fragments, can include a cytoxic agent that is conjugated to the antibody or fragment. In one aspect, the cytotoxic agent is monomethylauristatin-E (MMAE), however, other cytoxic agents are also provided, which can include, for example, functional analogs of MMAE (e.g. monomethylauristatin-F), and other cytotoxic agents, e.g., aplidin, azaribine, anastrozole, azacytidine, bleomycin, bortezomib, bryostatin-1, busulfan, calicheamycin, camptothecin, 10-hydroxycamptothecin, carmustine, celebrex, chlorambucil, cisplatin, irinotecan (CPT-I 1), SN-38, carboplatin, cladribine, cyclophosphamide, cytarabine, dacarbazine, docetaxel, dactiπomycin, daunomycin glucuronide, daunorubicin, dexamethasone, diethylstilbestrol, doxorubicin, doxorubicin glucuronide, epirubicin glucuronide, ethinyl estradiol, estramustine, etoposide, etoposide glucuronide, etoposide phosphate, floxuridine (FUdR), 3′,5′-O-dioleoyl-FUdR (FUdR-dO), fludarabine, flutamide, fluorouracil, fluoxymesterone, gemcitabine, hydroxyprogesterone caproate, hydroxyurea, idarubicin, ifosfamide, L-asparaginase, leucovorin, lomustine, mechlorethamine, medroprogesterone acetate, megestrol acetate, melphalan, mercaptopurine, 6-mercaptopurine, methotrexate, mitoxantrone, mithramycin, mitomycin, mitotane, phenyl butyrate, prednisone, procarbazine, paclitaxel, pentostatin, PSI-341, semustine streptozocin, tamoxifen, taxanes, taxol, testosterone propionate, thalidomide, thioguanine, thiotepa, teniposide, topotecan, uracil mustard, velcade, vinblastine, vinorelbine, vincristine, ricin, abrin, ribomiclease, onconase, rapLRl, DNase I, Staphylococcal enterotoxin-A, pokeweed antiviral protein, gelonin, diphtheria toxin, Pseudomonas exotoxin, and Pseudomonas endotoxin, or combinations thereof. Any of the cytoxic agents can also include functional analogs thereof.

Antibody Technology

A number of technologies are available to produce antibodies. For example, phage-antibody technology may be used to generate antibodies (Knappik, et al., J. Mol. Biol. 296:57-86, 2000). Another approach for obtaining antibodies is to screen a DNA library from B cells as described by Dower, et al., (WO 91/17271) and McCafferty, et al., (WO 92/01047). In these methods, libraries of phage are produced in which members display different antibodies on their outer surfaces. Antibodies are usually displayed as Fv or Fab fragments. Phage displaying antibodies are selected by affinity enrichment for binding to a selected protein. Antibodies may also be produced using trioma methodology (Oestberg, et al., Hybridoma 2:361-367, 1983; U.S. Pat. No. 4,634,664; U.S. Pat. No. 4,634,666).

Antibodies may also be purified from any cell that expresses the antibodies, including host cells that have been transfected with antibody-encoding expression constructs. The host cells may be cultured under conditions whereby the antibodies are expressed. Purified antibody may be separated from other cellular components that may associate with the antibody in the cell, such as certain proteins, carbohydrates, or lipids using methods well known in the art. Such methods include, but are not limited to, size exclusion chromatography, ammonium sulfate fractionation, ion exchange chromatography, affinity chromatography, and preparative gel electrophoresis. Purity of the preparations may be assessed by any means known in the art, such as SDS-polyacrylamide gel electrophoresis. A preparation of purified antibodies may contain more than one type of antibody.

Alternatively, antibodies may be produced using chemical methods to synthesize its amino acid sequence, such as by direct peptide synthesis using solid-phase techniques (see, e.g., Merrifield, J. Am. Chem. Soc. 85:2149-2154, 1963; Roberge, et al., Science 269:202-204, 1995). Protein synthesis may be performed using manual techniques or by automation. Automated synthesis may be achieved, for example, using Applied Biosystems 431A Peptide Synthesizer (Perkin Elmer). Optionally, fragments of antibodies may be separately synthesized and combined using chemical methods to produce a full-length molecule.

The antibodies of the present invention may be generated from parental antibodies. Parent antibodies may be selected from various antibodies capable of binding specific targets and well known in the art, such as, but not limited to, but are not limited to anti-TNF antibody, anti-IL-12 antibody; anti-IL-18 antibody, anti-05, anti-CD147, anti-gp120, anti-CD11a, anti-CD18, anti-VEGF, anti-CD40L, anti-ICAM-1, anti-CD2, anti-EGFR, anti-TGF-beta 2, anti-E-selectin, anti-Her2/neu, anti-CD14, anti-ICAM-3, anti-CD80, anti-CD4, anti-CD3, anti-CD23, anti-beta2-integrin, anti-CD52, anti-CD22, anti-CD20, anti-CD25, anti-CD33, anti-HLA, anti-IL-1alpha, anti-IL-1, anti-IL-1 receptor, anti-IL-2 receptor, anti-IL-4, anti-IL4 receptor, anti-IL5, anti-IL-5 receptor, anti-IL-6, anti-IL-8, anti-IL-9, anti-IL-13, anti-IL-13 receptor, anti-IL-17, and anti-IL-23. Parent antibodies may also be selected from various therapeutic antibodies including, but are not limited to, rituximab, trastuzumab, pertuzumab, cetuximab, alemtuzumab, muromonab, ibritumomab, gemtuzumab ozogamicin, alefacept, abciximab, basiliximab, palivizumab, infliximab, adalimumab, etanercept, natalizumab, bevacizumab, omalizumab, efalizumab, clenoliximab, labetuzumab, epratuzumab, and visilizumab.

The newly synthesized molecules may be substantially purified by preparative high performance liquid chromatography (see, e.g., Creighton, Proteins: Structures and Molecular Principles, WH Freeman and Co., New York, N.Y., 1983). The composition of a synthetic polypeptide may be confirmed by amino acid analysis or sequencing (e.g., using Edman degradation).

The present invention also relates to bispecific or bifunctional antibodies that have one binding site that specifically binds to a first antigen and a second binding site that specifically binds to a second antigen. This results in multi-functional valency, that is, an ability to bind at least two different epitopes simultaneously.

Polynucleotides Encoding Antibodies

The present invention also relates to polynucleotides encoding antibodies. These polynucleotides may be used, for example, to produce quantities of the antibodies for therapeutic or diagnostic use.

Polynucleotides of the present invention may also be isolated from host cells, free of other cellular components such as membrane components, proteins, and lipids. Polynucleotides may be isolated using standard nucleic acid purification techniques, or synthesized using an amplification technique such as the polymerase chain reaction (PCR), or by using an automatic synthesizer. Methods for isolating polynucleotides are routine and are known in the art. Any such technique for obtaining a polynucleotide may be used to obtain isolated polynucleotides encoding antibodies of the invention. For example, restriction enzymes and probes may be used to isolate polynucleotides which encode antibodies.

Antibody-encoding cDNA molecules may be made with standard molecular biology techniques, using mRNA as a template. Thereafter, cDNA molecules may be replicated using molecular biology techniques known in the art and disclosed in manuals such as Sambrook, et al., (Molecular Cloning: A Laboratory Manual, (Second Edition, Cold Spring Harbor Laboratory Press; Cold Spring Harbor, N.Y.; 1989) Vol. 1-3). An amplification technique, such as PCR, may be used to obtain additional copies of the polynucleotides. Alternatively, synthetic chemistry techniques may be used to synthesize polynucleotides encoding antibodies of the invention.

To express a polynucleotide encoding an antibody, the polynucleotide may be inserted into an expression vector that contains the necessary elements for the transcription and translation of the inserted coding sequence. Methods that are well known to those skilled in the art may be used to construct expression vectors containing sequences encoding antibodies and appropriate transcriptional and translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Such techniques are described, for example, in Sambrook, et al. (1989) and in Ausubel, et al., (Current Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y., 1995).

A variety of expression vector/host systems may be utilized to contain and express sequences encoding antibodies. These include, but are not limited to, microorganisms, such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with virus expression vectors (e.g., baculovirus); plant cell systems transformed with virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV); or bacterial expression vectors (e.g., Ti or pBR322 plasmids), or animal cell systems.

The control elements or regulatory sequences are those non-translated regions of the vector—enhancers, promoters, 5′ and 3′ untranslated regions—which interact with host cellular proteins to carry out transcription and translation. Such elements may vary in strength and specificity. Depending on the vector system and host utilized, any number of suitable transcription and translation elements, including constitutive and inducible promoters, may be used. For example, when cloning in bacterial systems, inducible promoters can be used. The baculovirus polyhedrin promoter may be used in insect cells. Promoters or enhancers derived from the genomes of plant cells (e.g., heat shock, RUBISCO, and storage protein genes) or from plant viruses (e.g., viral promoters or leader sequences) may be cloned into the vector. In mammalian cell systems, promoters from mammalian genes or from mammalian viruses may be used. If it is necessary to generate a cell line that contains multiple copies of a nucleotide sequence encoding an antibody, vectors based on SV40 or EBV may be used with an appropriate selectable marker.

General texts describing additional molecular biological techniques useful herein, including the preparation of antibodies include Berger and Kimmel (Guide to Molecular Cloning Techniques, Methods in Enzymology, Vol. 152, Academic Press, Inc.); Sambrook, et al., (Molecular Cloning: A Laboratory Manual, (Second Edition, Cold Spring Harbor Laboratory Press; Cold Spring Harbor, N.Y.; 1989) Vol. 1-3); Current Protocols in Molecular Biology, (F. M. Ausabel et al. [Eds.], Current Protocols, a joint venture between Green Publishing Associates, Inc. and John Wiley & Sons, Inc. (supplemented through 2000)); Harlow et al., (Monoclonal Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1988), Paul [Ed.]); Fundamental Immunology, (Lippincott Williams & Wilkins (1998)); and Harlow, et al., (Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1998)).

Assays

The affinity (K_(d)) of antibody binding to an antigen may be assayed using any method known in the art including, for example, immunoassays such as enzyme-linked immununospecific assay (ELISA), Bimolecular Interaction Analysis (BIA) (see, e.g., Sjolander and Urbaniczky; Anal. Chem. 63:2338-2345, 1991; Szabo, et al., Curr. Opin. Struct. Biol. 5:699-705, 1995), and fluorescence-activated cell sorting (FACS) for quantification of antibody binding to cells that express an antigen. BIA is a technology for analyzing biospecific interactions in real time, without labeling any of the interactants (e.g., BIAcore™). Changes in the optical phenomenon surface plasmon resonance (SPR) may be used as an indication of real-time reactions between biological molecules.

The present invention also relates to the use of quantitative immunoassays to measure levels of proteins in patient samples. Many formats may be adapted for use with the methods of the present invention. For example, the detection and quantitation of a protein in patient samples may be performed, by enzyme-linked immunosorbent assays, radioimmunoassays, dual antibody sandwich assays, agglutination assays, fluorescent immunoassays, immunoelectron and scanning microscopy, among other assays commonly known in the art. The quantitation of a protein in such assays may be adapted by conventional methods known in the art. Serial changes in circulating a protein levels may be detected and quantified by a sandwich assay in which the capture antibody has been immobilized using conventional techniques on the surface of the support.

Suitable supports include, for example, synthetic polymer supports, such as polypropylene, polystyrene, substituted polystyrene, polyacrylamides (such as polyamides and polyvinylchloride), glass beads, agarose, and nitrocellulose.

The antibodies useful to identify proteins may be labeled in any conventional manner. An example of a label is horseradish peroxidase, and an example of a method of labeling antibodies is by using biotin-strepavidin complexes.

As appropriate, antibodies used in the immunoassays of this invention that are used as tracers may be labeled in any manner, directly or indirectly, that results in a signal that is visible or can be rendered visible. Detectable marker substances include radionuclides, such as ³H, ¹²⁵I, and ¹³¹I; fluorescers, such as, fluorescein isothiocyanate and other fluorochromes, phycobiliproteins, phycoerythin, rare earth chelates, Texas red, dansyl and rhodamine; colorimetric reagents (chromogens); electron-opaque materials, such as colloidal gold; bioluminescers; chemiluminescers; dyes; enzymes, such as, horseradish peroxidase, alkaline phosphatases, glucose oxidase, glucose-6-phosphate dehydrogenase, acetylcholinesterase, alpha beta-galactosidase, among others; coenzymes; enzyme substrates; enzyme cofactors; enzyme inhibitors; enzyme subunits; metal ions; free radicals; or any other immunologically active or inert substance which provides a means of detecting or measuring the presence or amount of immunocomplex formed. Exemplary of enzyme substrate combinations are horseradish peroxidase and tetramethyl benzidine (TMB), and alkaline phosphatases and paranitrophenyl phosphate (pNPP).

Another detection and quantitation systems according to this invention produce luminescent signals, bioluminescent (BL) or chemiluminescent (CL). In chemiluminescent (CL) or bioluminescent (BL) assays, the intensity or the total light emission is measured and related to the concentration of the unknown analyte. Light can be measured quantitatively using a luminometer (photomultiplier tube as the detector) or charge-coupled device, or qualitatively by means of photographic or X-ray film. The main advantages of using such assays is their simplicity and analytical sensitivity, enabling the detection and/or quantitation of very small amounts of analyte.

Exemplary luminescent labels are acridinium esters, acridinium sulfonyl carboxamides, luminol, umbelliferone, isoluminol derivatives, photoproteins, such as aequorin, and luciferases from fireflies, marine bacteria, Vargulla and Renilla. Luminol can be used optionally with an enhancer molecule such as 4-iodophenol or 4-hydroxy-cinnamic acid. Typically, a CL signal is generated by treatment with an oxidant under basic conditions.

Additional luminescent detection systems are those wherein the signal (detectable marker) is produced by an enzymatic reaction upon a substrate. CL and BL detection schemes have been developed for assaying alkaline phosphatases (AP), glucose oxidase, glucose 6-phosphate dehydrogenase, horseradish peroxidase (HRP), and xanthine-oxidase labels, among others. AP and HRP are two enzyme labels which can be quantitated by a range of CL and BL reactions. For example, AP can be used with a substrate, such as an adamantyl 1,2-dioxetane aryl phosphate substrate (e.g. AMPPD or CSPD; Kricka, L. J., “Chemiluminescence and Bioluminescence, Analysis by,” Molecular Biology and Biotechnology: A Comprehensive Desk Reference (ed. R. A. Meyers) (VCH Publishers; N.Y., N.Y.; 1995)); for example, a disodium salt of 4-methoxy-4-(3-phosphatephenyl)spiro[1,2-dioxetane-3,2′-adamantane], with or without an enhancer molecule such as 1-(trioctylphosphonium methyl)-4-(tributylphosphonium methyl)benzene diochloride. HRP is may be used with substrates, such as, 2′,3′,6′-trifluorophenyl-methoxy-10-methylacridan-9-carboxylate.

CL and BL reactions may be adapted for analysis not only of enzymes, but also of other substrates, cofactors, inhibitors, metal ions, and the like. For example, luminol, firefly luciferase, and marine bacterial luciferase reactions are indicator reactions for the production or consumption of peroxide, ATP, and NADPH, respectively. They may be coupled to other reactions involving oxidases, kinases, and dehydrogenases, and may be used to measure any component of the coupled reaction (enzyme, substrate, cofactor).

The detectable marker may be directly or indirectly linked to an antibody used in an assay of this invention. Exemplary of an indirect linkage of the detectable label is the use of a binding pair between an antibody and a marker or the use of a signal amplification system.

Examples of binding pairs that may be used to link antibodies to detectable markers are biotin/avidin, streptavidin, or anti-biotin; avidin/anti-avidin; thyroxine/thyroxine-binding globulin; antigen/antibody; antibody/ anti-antibody; carbohydrate/lectins; hapten/anti-hapten antibody; dyes and hydrophobic molecules/hydrophobic protein binding sites; enzyme inhibitor, coenzyme or cofactor/enzyme; polynucleic acid/homologous polynucleic acid sequence; fluorescein/anti-fluorescein; dinitrophenollanti-dinitrophenol; vitamin B 12/intrinsic factor; cortisone, cortisol/cortisol binding protein; and ligands for specific receptor protein/membrane associated specific receptor proteins.

Various means for linking labels directly or indirectly to antibodies are known in the art. For example, labels may be bound either covalently or non-covalently. Exemplary antibody conjugation methods are described in Avarmeas, et al., Scan. J. Immunol. 8(Suppl. 7): 7, 1978); Bayer, et al., Meth. Enzymol. 62:308, 1979; Chandler, et al., J. Immunol. Meth. 53:187, 1982; Ekeke and Abuknesha, J. Steroid Biochem. 11:1579, 1979; Engvall and Perlmann, J. Immunol. 109:129, 1972; Geoghegan, et al., Immunol. Comm. 7:1, 1978; and Wilson and Nakane, Immunofluorescence and Related Techniques, Elsevier/North Holland Biomedical Press; Amsterdam (1978).

Depending upon the nature of the label, various techniques may be employed for detecting and quantitating the label. For fluorescers, a large number of fluorometers are available. For chemiluminescers, luminometers or films are available. With enzymes, a fluorescent, chemiluminescent, or colored product may be determined or measured fluorometrically, luminometrically, spectrophotometrically, or visually.

Various types of chemiluminescent compounds having an acridinium, benzacridinium, or acridan type of heterocyclic ring systems are other examples of labels. Examples of acridinium esters include those compounds having heterocyclic rings or ring systems that contain the heteroatom in a positive oxidation state including such ring systems as acridinium, benz[a]acridinium, benz[b]acridinium, benz[c]acridinium, a benzimidazole cation, quinolinium, isoquinolinium, quinolizinium, a cyclic substituted quinolinium, phenanthridinium, and quinoxalinium.

The tracer may be prepared by attaching to the selected antibody either directly or indirectly a reactive functional group present on the acridinium or benzacridinium ester, as is well known to those skilled in the art (see, e.g., Weeks, et al., Clin. Chem. 29(8):1474-1479, 1983). Examples of compounds are acridinium and benzacridinium esters with an aryl ring leaving group and the reactive functional group present in either the para or the meta position of the aryl ring. (see, e.g., U.S. Pat. No. 4,745,181 and WO 94/21823).

Methods of Use

As used herein, various terms are defined below.

The term “treatment” includes any process, action, application, therapy, or the like, wherein a subject (or patient), including a human being, is provided medical aid with the object of improving the subject's condition, directly or indirectly, or slowing the progression of a condition or disorder in the subject.

The term “combination therapy” or “co-therapy” means the administration of two or more therapeutic agents to treat a disease, condition, and/or disorder. Such administration encompasses co-administration of two or more therapeutic agents in a substantially simultaneous manner, such as in a single capsule having a fixed ratio of active ingredients or in multiple, separate capsules for each inhibitor agent. In addition, such administration encompasses use of each type of therapeutic agent in a sequential manner.

The antibodies of the invention may be administered in combination with the following agents: cytotoxic agent, angiogenesis inhibitors, antirheumatic agent, muscle relaxant, narcotic, non-steroid anti-inflammatory drug, analgesic, anesthetic, sedative, local anesthetic, neuromuscular blocker, antimicrobial agent, immunoglobulins, antidepressant, asthma medication, cytokine, and cytokine antagonist.

For example, the antibodies of the invention may be administered in combination with various anti-cancer agents including, but not limited to, bleomycin, docetaxel, doxorubicin, edatrexate, erlotinib, etoposide, finasteride, flutamide, gemcitabine, genitinib, goserelin acetate, granisetron, imatinib, irinotecan, ondansetron, paclitaxel, pegaspargase, pilocarpine hydrochloride, porfimer sodium, interleukin-2, rituximab, topotecan, trastuzumab, triapine, vincristine, and vinorelbine tartrate, or therapeutic antibodies or fragments thereof, or anti-angiogenic agent, such as, for example, angiostatin, bevacizumab, sorafenib, baculostatin, canstatin, maspin, anti-VEGF antibodies or peptides, anti-placental growth factor antibodies or peptides, anti-Flk-1 antibodies, anti-Fit-1 antibodies or peptides, laminin peptides, fibronectin peptides, plasminogen activator inhibitors, tissue metalloproteinase inhibitors, interferons, interleukin 12, Gro-β, thrombospondin, 2-methoxyoestradiol, proliferin-related protein, carboxiamidotriazole, CM1O1, Marimastat, pentosan polysulphate, angiopoietin 2, interferon-alpha, herbimycin A, PNU145156E, 16K prolactin fragment, Linomide, thalidomide, pentoxifylline, genistein, TNP-470, endostatin, paclitaxel, accutin, cidofovir, vincristine, bleomycin, AGM-1470, platelet factor 4 or minocycline.

The phrase “therapeutically effective” means the amount of each agent administered that will achieve the goal of improvement in a disease, condition, and/or disorder severity, while avoiding or minimizing adverse side effects associated with the given therapeutic treatment.

The term “pharmaceutically acceptable” means that the subject item is appropriate for use in a pharmaceutical product.

The antibodies of this invention are expected to be valuable as therapeutic agents. Accordingly, an embodiment of this invention includes a method of treating the various conditions in a patient (including mammals) which comprises administering to said patient a composition containing an amount of an antibody of the invention that is effective in treating the target condition.

The antibodies of the present invention may be used in the treatment or prevention of various diseases including, but not limited to, cancer, infectious disease, and autoimmune diseases.

The antibodies of the present invention or compositions including the antibodies may include a cytotoxic agent (e.g., monomethylauristatin-E) that is conjugated to the antibody.

Antibodies of the present invention may be administered alone or in combination with one or more additional therapeutic agents. Combination therapy includes administration of a single pharmaceutical dosage formulation which contains an antibody of the present invention and one or more additional therapeutic agents, as well as administration of the antibody of the present invention and each additional therapeutic agents in its own separate pharmaceutical dosage formulation. For example, an antibody of the present invention and a therapeutic agent may be administered to the patient together in a single oral dosage composition or each agent may be administered in separate oral dosage formulations.

Where separate dosage formulations are used, the antibody of the present invention and one or more additional therapeutic agents may be administered at essentially the same time (e.g., concurrently) or at separately staggered times (e.g., sequentially).

To assess the ability of a particular antibody to be therapeutically useful to treat cancer, as an example, the antibody may be tested in vivo in a mouse xenograft tumor model. An example of a therapeutic model is detailed in Example 8.

Pharmaceutical Compositions

The antibodies described herein may be provided in a pharmaceutical composition comprising a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may be non-pyrogenic. The compositions may be administered alone or in combination with at least one other agent, such as stabilizing compound, which may be administered in any sterile, biocompatible pharmaceutical carrier including, but not limited to, saline, buffered saline, dextrose, and water. A variety of aqueous carriers may be employed including, but not limited to saline, glycine, or the like. These solutions are sterile and generally free of particulate matter. These solutions may be sterilized by conventional, well known sterilization techniques (e.g., filtration). The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, and the like. The concentration of the antibody of the invention in such pharmaceutical formulation may vary widely, and may be selected primarily based on fluid volumes, viscosities, etc., according to the particular mode of administration selected. If desired, more than one type of antibody may be included in a pharmaceutical composition.

The compositions may be administered to a patient alone, or in combination with other agents, drugs or hormones. In addition to the active ingredients, these pharmaceutical compositions may contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries that facilitate processing of the active compounds into preparations which may be used pharmaceutically. Pharmaceutical compositions of the invention may be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, parenteral, topical, sublingual, or rectal means.

Formulations suitable for subcutaneous, intravenous, intramuscular, and the like; suitable pharmaceutical carriers; and techniques for formulation and administration may be prepared by any of the methods well known in the art (see, e.g., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 20^(th) edition, 2000).

Diagnostic Methods

The present invention also provides diagnostic methods with which a particular antigen may be detected in a patient sample or biological sample. Such diagnostic methods may be used, for example, to diagnose disorders in which a particular antigen is elevated or reduced. Such disorders include, but are not limited to, cancer, infectious disease, and autoimmune diseases. As an example, when used for diagnosis, detection of an amount of the antibody-antigen complex in a sample from a patient which is greater than an amount of the complex in a normal sample identifies the patient as likely to have the disorder

The patient sample may be contacted with an antibody of the invention, and the patient sample may then be assayed for the presence of an antibody-antigen complex. As described above, the antibody may comprise a detectable label, such as a fluorescent, radioisotopic, chemiluminescent, or enzymatic label, such as horseradish peroxidase, alkaline phosphatase, or luciferase.

Optionally, the antibody may be bound to a solid support, which may accommodate automation of the assay. Suitable solid supports include, but are not limited to, glass or plastic slides, tissue culture plates, microtiter wells, tubes, silicon chips, or particles such as beads (including, but not limited to, latex, polystyrene, or glass beads). Any method known in the art may be used to attach the antibody to the solid support, including use of covalent and non-covalent linkages, passive absorption, or pairs of binding moieties attached to the antibody and the solid support. Binding of antigen and the antibody may be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtiter plates, test tubes, and microcentrifuge tubes.

Determination of a Therapeutically Effective Dose

The determination of a therapeutically effective dose is well within the capability of those skilled in the art. A therapeutically effective dose refers to the amount of an antibody that may be used to effectively treat a disease (e.g., cancer) compared with the efficacy that is evident in the absence of the therapeutically effective dose.

The therapeutically effective dose may be estimated initially in animal models (e.g., rats, mice, rabbits, dogs, or pigs). The animal model may also be used to determine the appropriate concentration range and route of administration. Such information may then be used to determine useful doses and routes for administration in humans.

Therapeutic efficacy and toxicity (e.g., ED₅₀—the dose therapeutically effective in 50% of the population and LD₅₀—the dose lethal to 50% of the population) of an antibody may be determined by standard pharmaceutical procedures in cell cultures or experimental animals. The dose ratio of toxic to therapeutic effects is the therapeutic index, and it may be expressed as the ratio, LD₅₀/ED₅₀. The data obtained from animal studies may used in formulating a range of dosage for human use. The dosage contained in such compositions may be within a range of circulating concentrations that include the ED₅₀ with little or no toxicity. The dosage varies within this range depending upon the dosage form employed, sensitivity of the patient, and the route of administration.

The exact dosage may be determined by the practitioner, in light of factors related to the patient who requires treatment. Dosage and administration may be adjusted to provide sufficient levels of the antibody or to maintain the desired effect. Factors that may be taken into account include the severity of the disease state, general health of the subject, age, weight, and gender of the subject, diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy. Effective in vivo dosages of an antibody are in the range of about 5 μg to about 500 μg/kg of patient body weight.

The mode of administration of antibody-containing pharmaceutical compositions of the present invention may be any suitable route which delivers the antibody to the host. As an example, pharmaceutical compositions of the invention may be useful for parenteral administration (e.g., subcutaneous, intramuscular, intravenous, or intranasal administration).

All patents and patent applications cited in this disclosure are expressly incorporated herein by reference. The above disclosure generally describes the present invention. A more complete understanding can be obtained by reference to the following specific examples, which are provided for purposes of illustration only and are not intended to limit the scope of the invention.

EXAMPLES

In order that this invention may be better understood, the following examples are set forth. These examples are for the purpose of illustration only, and are not to be construed as limiting the scope of the invention in any manner. All publications mentioned herein are incorporated by reference in their entirety.

Example 1 Construction, Expression, and Purification of Fab-Like Antibody

Antibodies 3E10 and 19G9 recognize tissue factor (TF) and tumor-associated antigen RG1, respectively. These two antibodies were used to construct protease-regulated antibodies containing the protease site DDDDK (SEQ ID NO: 26) linker located between the antigen binding domains and constant region domain. Specifically, these antibodies contained V_(L)-DDDDK-CL on the light chain and V_(H)-DDDDK-CH1-Myc-His6 on the heavy chain, where the linker is cleavable by enterokinase, and Myc and His6 are tags for detection and purification. The DNA sequences for the two antibodies were cloned into bacterial expression vectors using standard molecular biology technologies, and the constructs were confirmed by DNA sequencing. Examples of plasmid are shown in FIGS. 8 and 9. The plasmid containing either 3E10 or 19G9 was expressed and purified from bacterial strain TG1. Briefly, a single colony of bacteria strain TG1 containing the antibody expression plasmid was selected and grown overnight in 8 ml of 2× YT medium in the presence of 34 μg/ml chloramphenicol and 1% glucose. A volume of culture (7 ml) was transferred to 250 ml fresh 2× YT medium containing 34 μg/ml chloramphenicol and 0.1% glucose. After 3 hours of incubation, 0.5 mM IPTG was added to induce Fab expression. The culture was incubated overnight at 25° C. Following incubation, the culture was centrifuged to pellet the bacterial cells, and the pellet was resuspended in a Bug Buster® lysis buffer (Novagen, Madison, Wis.). After centrifugation, the bacterial lysis supernatant was filtered, and the Fab fragments were affinity-purified through a Ni-NTA column (Qiagen, Valencia, Calif.) according to the manufacturer's instruction.

Other examples of protease-regulated antibodies were also constructed using tandem linked variable regions from 3E10 and 1909. These antibodies contained, for example, V_(L)3E10-DDDDK-V_(L)19G9-CL on the light chain and V_(H)3E10-DDDDK-V_(H)19G9-CH1-Myc-His6 on the heavy chain, where the linker is cleavable by enterokinase, and Myc and His6 are tags for detection and purification. An antibody library was also constructed using the framework regions (FR), for example, FR4 of 3E10 and FR1 of 19G9 either intact or truncated. Several types of protease-regulated antibodies were screened from this library. The cloning, expression, and purification were performed as described above.

Example 2 Cloning and Expression of IgG-Like Antibodies

The expression vector pIE_SRgamma_fa contains cDNAs encoding the constant regions of human IgGI (fa haplotype) and kappa chains, respectively. An overlap PCR was performed to link the variable regions of anti-TF antibody 3E10 and anti-RG1 antibody 19G9. The native signal peptide of 19G9 was used for secretion of the protease-regulated antibodies. Four examples of peptide linkers located between the variable regions of 3E10 and 19G9 are Linker 1: SDDDDK (SEQ ID NO: 2), Linker 2: GGGGSDDDDK (SEQ ID NO: 3), Linker 3: GGGGSDDDDKGGGGS (SEQ ID NO: 4), and Linker 4: GGGGSGGGGSGGGGS (SEQ ID NO: 5). The primers for amplification of the variable region of the light chain introduced Hind III and Bsiw I sites into the 5′ and 3′ ends of PCR fragment, respectively. The resulting PCR-amplified V_(L) genes were cloned into the HindIII/Bsiw site of pIE_SRgamma1_fa to create pIE-3E10V_(L)-linker-19G9V_(L). The same strategy was used to clone in frame V_(H) fusions of 3E10 and 19G9 (including linkers 1-4) into pIE-3E10V_(L)-linker-19G9V_(L). Briefly, the primer pairs of the variable regions of 3E10 and 19G9 contained NotI/ApaI sites. The PCR products were digested with NotI/ApaI and inserted upstream of the CH region of pIE-3E10V_(H)-linker-19G9V_(H) ensuring that the V_(H) regions were in frame with the CH region in the respective pIE derivatives. The final constructs were verified by DNA sequencing analysis.

Transfection and transient expression of the protease-regulated antibodies were conducted using mammalian cells. Approximately 4×10⁸ CHO-S cells supplemented with CHO-SF medium were prepared for transfection. Transfection was carried out using Lipofectamine™ 2000 (Invitrogen, Carlsbad, Calif.) and 1 mg plasmid DNA following the manufacturer's instruction. The cells were grown for three days after transfection, and the culture media was harvested and filtered for antibody isolation and purification.

Examples of the protease-regulated antibodies are described in Tables 3-9

TABLE 3 PROTEASE-REGULATED ANTIBODIES (Type 1) Light chain Heavy chain Fab-like protease-regulated antibodies against TF (3E10) DIVLTQPHSVSASPGKTVTISCTRSSGSVA DLVESGGTLVQPGGSLRLSCAASGFSFTDAW SYYVQWYQQRPGSSPTTVIYEDNHRPSGVP MSWVRQAPGKELEWVSSISGSGGSTYYAGSV DRFSGSIDTSSNSASLTISGLKTEDEADYY KGRFTISRDNSKNTLYLQMNSLRAEDTAVYY CQSYDSNNLVVFGGGTKLTVLGAGGGGSDD CARVLSLTDYYWYGMDVWGQGTLVTVSASDD DDKRTVAAPSVFIFPPSDEQLKSGTASVVC DDKSSASTKGPSVFPLAPSSKSTSGGTAALG LLNNFYPREAKVQWKVDNALQSGNSQESVT CLVKDYFPEPVTVSWNSGALTSGVHTFPAVL EQDSKDSTYSLSSTLTLSKADYEKHKVYAC QSSGLYSLSSVVTVPSSSLGTQTYICNVNHK EVTHQGLSSPVTKSFNRGEC PSNTKVDKKVEPKCEF (SEQ ID NO: 33) (SEQ ID NO: 34) Fab-like protease-regulated antibodies against RG1 (19G9) DIVLTQSPGTLSLSPGERATLSCRASQSVSS QLVQSGGGLVQPGGSLRLSCAGSGFTFSSYV SYLAWYQQKPGQAPRLLIYGASSRATGIPDR MHWLRQAPGKGLEWVSVIGTGGVTHYADSVK FSGSGSGTDFTLTISRLEPEDFAVYYCQQYS GRFTISRDNAKNSLYLQMNSLRAEDTAVYYC SSLTFGGGTKVEIKDDDDKRTVAAPSVFIFP ARWGYYGSGSYENDAFDIWGQGTMVTVDDDD PSDEQLKSGTASVVCLLNNFYPREAKVQWKV KSSASTKGPSVFPLAPSSKSTSGGTAALGCL DNALQSGNSQESVTEQDSKDSTYSLSSTLTL VKDYFPEPVTVSWNSGALTSGVHTFPAVLQS SKADYEKHKVYACEVTHQGLSSPVTKSFNRG SGLYSLSSVVTVPSSSLGTQTYICNVNHKPS EC NTKVDKKVEPKCEF (SEQ ID NO: 35) (SEQ ID NO: 36) IgG-like protease-regulated antibodies against TF (3E10) NFMLTQPHSVSASPGKTVTISCTRSSGSVAS QVNLRESGGTLVQPGGSLRLSCAASGFSFTD YYVQWYQQRPGSSPTTVIYEDNHRPSGVPDR AWMSWVRQAPGKELEWVSSISGSGGSTYYAG FSGSIDTSSNSASLTISGLKTEDEADYYCQS SVKGRFTISRDNSKNTLYLQMNSLRAEDTAV YDSNNLVVFGGGTKLTVLGQSDDDDKPKAAP YYCARVLSLTDYYWYGMDVWGQGTLVTVSAS SVTLFPPSSEELQANKATLVCLISDFYPGAV DDDDKTKGPSVFPLAPSSKSTSGGTAALGCL TVAWKADSSPVKAGVETTTPSKQSNNKYAAS VKDYFPEPVTVSWNSGALTSGVHTFPAVLQS SYLSLTPEQWKSHRSYSCQVTHEGSTVEKTV SGLYSLSSVVTVPSSSLGTQTYICNVNHKPS APTECS NTKVDKRVEPKSCDKTHTCPPCPAPELLGGP (SEQ ID NO: 37) SVFLFPPKPKDTLMISRTPEVTCVVVDVSHE DPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKALPAP IEKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS CSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 38) IgG-like protease-regulated antibodies against RG1 (19G9) EIVLTQSPGTLSLSPGERATLSCRASQSVS EVQLVQSGGGLVQPGGSLRLSCAGSGFTFS SSYLAWYQQKPGQAPRLLIYGASSRATGIP SYVMHWLRQAPGKGLEWVSVIGTGGVTHYA DRFSGSGSGTDFTLTISRLEPEDFAVYYCQ DSVKGRFTISRDNAKNSLYLQMNSLRAEDT QYSSSLTFGGGTKVEIKRTSDDDDKVAAPS AVYYCARWGYYGSGSYENDAFDIWGQGTMV VFIFPPSDEQLKSGTASVVCLLNNFYPREA TVSSASDDDDDKTKGPSVFPLAPSSKSTSG KVQWKVDNALQSGNSQESVTEQDSKDSTYS GTAALGCLVKDYFPEPVTVSWNSGALTSGV LSSTLTLSKADYEKHKVYACEVTHQGLSSP HTFPAVLQSSGLYSLSSVVTVPSSSLGTQT VTKSFNRGEC YICNVNHKPSNTKVDKRVEPKSCDKTHTCP (SEQ ID NO: 39) PCPAPELLGGPSVFLFPPKPKDTLMISRTP EVTCVVVDVSHEDPEVKFNWYVDGVEVHNA KTKPREEQYNSTYRVVSVLTVLHQDWLNGK EYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSREEMTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGK (SEQ ID NO: 40)

TABLE 4 PROTEASE-REGULATED ANTIBODIES (Type 2) Fab-like protease-regulated antibodies against TF and RG1 Light chain Heavy chain H1L1 DIVLTQPHSVSASPGKTVTISCTRSSGSVA QVQLVESGGTLVQPGGSLRLSCAASGFSFT SYYVQWYQQRPGSSPTTVIYEDNHRPSGVP DAWMSWVRQAPGKELEWVSSISGSGGSTYY DRFSGSIDTSSNSASLTISGLKTEDEADYY AGSVKGRFTISRDNSKNTLYLQMNSLRAED CQSYDSNNLVVFGGGTKLTVLGASDDDDKE TAVYYCARVLSLTDYYWYGMDVWGQGTLVT IVLTQSPGTLSLSPGERATLSCRASQSVSS VSASDDDDKEVQLVQSGGGLVQPGGSLRLS SYLAWYQQKPGQAPRLLIYGASSRATGIPD CAGSGFTFSSYVMHWLRQAPGKGLEWVSVI RFSGSGSGTDFTLTISRLEPEDFAVYYCQQ GTGGVTHYADSVKGRFTISRDNAKNSLYLQ YSSSLTFGGGTKVEIKRTVAAPSVFIFPPS MNSLRAEDTAVYYCARWGYYGSGSYENDAF DEQLKSGTASVVCLLNNFYPREAKVQWKVD DIWGQGTMVTVSSASTKGPSVFPLAPSSKS NALQSGNSQESVTEQDSKDSTYSLSSTLTL TSGGTAALGCLVKDYFPEPVTVSWNSGALT SKADYEKHKVYACEVTHQGLSSPVTKSFNR SGVHTFPAVLQSSGLYSLSSVVTVPSSSLG GEC TQTYICNVNHKPSNTKVDKKVEPKCEF (SEQ ID NO: 41) (SEQ ID NO: 42) H1L4 DIVLTQPHSVSASPGKTVTISCTRSSGSVA QVQLVESGGTLVQPGGSLRLSCAASGFSFT SYYVQWYQQRPGSSPTTVIYEDNHRPSGVP DAWMSWVRQAPGKELEWVSSISGSGGSTYY DRFSGSIDTSSNSASLTISGLKTEDEADYY AGSVKGRFTISRDNSKNTLYLQMNSLRAED CQSYDSNNLVVFGGGTKLTVLGASDDDDKL TAVYYCARVLSLTDYYWYGMDVWGQGTLVT TQSPGTLSLSPGERATLSCRASQSVSSSYL VSASDDDDKEVQLVQSGGGLVQPGGSLRLS AWYQQKPGQAPRLLIYGASSRATGIPDRFS CAGSGFTFSSYVMHWLRQAPGKGLEWVSVI GSGSGTDFTLTISRLEPEDFAVYYCQQYSS GTGGVTHYADSVKGRFTISRDNAKNSLYLQ SLTFGGGTKVEIKRTVAAPSVFIFPPSDEQ MNSLRAEDTAVYYCARWGYYGSGSYENDAF LKSGTASVVCLLNNFYPREAKVQWKVDNAL DIWGQGTMVTVSSASTKGPSVFPLAPSSKS QSGNSQESVTEQDSKDSTYSLSSTLTLSKA TSGGTAALGCLVKDYFPEPVTVSWNSGALT DYEKHKVYACEVTHQGLSSPVTKSFNRGEC SGVHTFPAVLQSSGLYSLSSVVTVPSSSLG (SEQ ID NO: 43) TQTYICNVNHKPSNTKVDKKVEPKCEF (SEQ ID NO: 44) H1L7 DIVLTQPHSVSASPGKTVTISCTRSSGSVA QVQLVESGGTLVQPGGSLRLSCAASGFSFT SYYVQWYQQRPGSSPTTVIYEDNHRPSGVP DAWMSWVRQAPGKELEWVSSISGSGGSTYY DRFSGSIDTSSNSASLTISGLKTEDEADYY AGSVKGRFTISRDNSKNTLYLQMNSLRAED CQSYDSNNLVVFGGGTKLTVLGASDDDDKS TAVYYCARVLSLTDYYWYGMDVWGQGTLVT PGTLSLSPGERATLSCRASQSVSSSYLAWY VSASDDDDKEVQLVQSGGGLVQPGGSLRLS QQKPGQAPRLLIYGASSRATGIPDRFSGSG CAGSGFTFSSYVMHWLRQAPGKGLEWVSVI SGTDFTLTISRLEPEDFAVYYCQQYSSSLT GTGGVTHYADSVKGRFTISRDNAKNSLYLQ FGGGTKVEIKRTVAAPSVFIFPPSDEQLKS MNSLRAEDTAVYYCARWGYYGSGSYENDAF GTASVVCLLNNFYPREAKVQWKVDNALQSG DIWGQGTMVTVSSASTKGPSVFPLAPSSKS NSQESVTEQDSKDSTYSLSSTLTLSKADYE TSGGTAALGCLVKDYFPEPVTVSWNSGALT KHKVYACEVTHQGLSSPVTKSFNRGEC SGVHTFPAVLQSSGLYSLSSVVTVPSSSLG (SEQ ID NO: 45) TQTYICNVNHKPSNTKVDKKVEPKCEF (SEQ ID NO: 46) H4L2 DIVLTQPHSVSASPGKTVTISCTRSSGSVA QVQLVESGGTLVQPGGSLRLSCAASGFSFT SYYVQWYQQRPGSSPTTVIYEDNHRPSGVP DAWMSWVRQAPGKELEWVSSISGSGGSTYY DRFSGSIDTSSNSASLTISGLKTEDEADYY AGSVKGRFTISRDNSKNTLYLQMNSLRAED CQSYDSNNLVVFGGGTKLTVLGDDDDKEIV TAVYYCARVLSLTDYYWYGMDVWGQGTLVT LTQSPGTLSLSPGERATLSCRASQSVSSSY VDDDDKQSGGGLVQPGGSLRLSCAGSGFTF LAWYQQKPGQAPRLLIYGASSRATGIPDRF SSYVMHWLRQAPGKGLEWVSVIGTGGVTHY SGSGSGTDFTLTISRLEPEDFAVYYCQQYS ADSVKGRFTISRDNAKNSLYLQMNSLRAED SSLTFGGGTKVEIKRTVAAPSVFIFPPSDE TAVYYCARWGYYGSGSYENDAFDIWGQGTM QLKSGTASVVCLLNNFYPREAKVQWKVDNA VTVSSASTKGPSVFPLAPSSKSTSGGTAAL LQSGNSQESVTEQDSKDSTYSLSSTLTLSK GCLVKDYFPEPVTVSWNSGALTSGVHTFPA ADYEKHKVYACEVTHQGLSSPVTKSFNRGE VLQSSGLYSLSSVVTVPSSSLGTQTYICNV C NHKPSNTKVDKKVEPKCEF (SEQ ID NO: 47) (SEQ ID NO: 48) H4L5 DIVLTQPHSVSASPGKTVTISCTRSSGSVA QVQLVESGGTLVQPGGSLRLSCAASGFSFT SYYVQWYQQRPGSSPTTVIYEDNHRPSGVP DAWMSWVRQAPGKELEWVSSISGSGGSTYY DRFSGSIDTSSNSASLTISGLKTEDEADYY AGSVKGRFTISRDNSKNTLYLQMNSLRAED CQSYDSNNLVVFGGGTKLTVLGDDDDKLTQ TAVYYCARVLSLTDYYWYGMDVWGQGTLVT SPGTLSLSPGERATLSCRASQSVSSSYLAW VDDDDKQSGGGLVQPGGSLRLSCAGSGFTF YQQKPGQAPRLLIYGASSRATGIPDRFSGS SSYVMHWLRQAPGKGLEWVSVIGTGGVTHY GSGTDFTLTISRLEPEDFAVYYCQQYSSSL ADSVKGRFTISRDNAKNSLYLQMNSLRAED TFGGGTKVEIKRTVAAPSVFIFPPSDEQLK TAVYYCARWGYYGSGSYENDAFDIWGQGTM SGTASVVCLLNNFYPREAKVQWKVDNALQS VTVSSASTKGPSVFPLAPSSKSTSGGTAAL GNSQESVTEQDSKDSTYSLSSTLTLSKADY GCLVKDYFPEPVTVSWNSGALTSGVHTFPA EKHKVYACEVTHQGLSSPVTKSFNRGEC VLQSSGLYSLSSVVTVPSSSLGTQTYICNV (SEQ ID NO: 49) NHKPSNTKVDKKVEPKCEF (SEQ ID NO: 50) H4L7 DIVLTQPHSVSASPGKTVTISCTRSSGSVA QVQLVESGGTLVQPGGSLRLSCAASGFSFT SYYVQWYQQRPGSSPTTVIYEDNHRPSGVP DAWMSWVRQAPGKELEWVSSISGSGGSTYY DRFSGSIDTSSNSASLTISGLKTEDEADYY AGSVKGRFTISRDNSKNTLYLQMNSLRAED CQSYDSNNLVVFGGGTKLTVLGASDDDDKS TAVYYCARVLSLTDYYWYGMDVWGQGTLVT PGTLSLSPGERATLSCRASQSVSSSYLAWY VDDDDKQSGGGLVQPGGSLRLSCAGSGFTF QQKPGQAPRLLIYGASSRATGIPDRFSGSG SSYVMHWLRQAPGKGLEWVSVIGTGGVTHY SGTDFTLTISRLEPEDFAVYYCQQYSSSLT ADSVKGRFTISRDNAKNSLYLQMNSLRAED FGGGTKVEIKRTVAAPSVFIFPPSDEQLKS TAVYYCARWGYYGSGSYENDAFDIWGQGTM GTASVVCLLNNFYPREAKVQWKVDNALQSG VTVSSASTKGPSVFPLAPSSKSTSGGTAAL NSQESVTEQDSKDSTYSLSSTLTLSKADYE GCLVKDYFPEPVTVSWNSGALTSGVHTFPA KHKVYACEVTHQGLSSPVTKSFNRGEC VLQSSGLYSLSSVVTVPSSSLGTQTYICNV (SEQ ID NO: 51) NHKPSNTKVDKKVEPKCEF (SEQ ID NO: 52) H5L5 DIVLTQPHSVSASPGKTVTISCTRSSGSVA QVQLVESGGTLVQPGGSLRLSCAASGFSFT SYYVQWYQQRPGSSPTTVIYEDNHRPSGVP DAWMSWVRQAPGKELEWVSSISGSGGSTYY DRFSGSIDTSSNSASLTISGLKTEDEADYY AGSVKGRFTISRDNSKNTLYLQMNSLRAED CQSYDSNNLVVFGGGTKLTVLGDDDDKLTQ TAVYYCARVLSLTDYYWYGMDVWGQGTLVT SPGTLSLSPGERATLSCRASQSVSSSYLAW VSASDDDDKLVQPGGSLRLSCAGSGFTFSS YQQKPGQAPRLLIYGASSRATGIPDRFSGS YVMHWLRQAPGKGLEWVSVIGTGGVTHYAD GSGTDFTLTISRLEPEDFAVYYCQQYSSSL SVKGRFTISRDNAKNSLYLQMNSLRAEDTA TFGGGTKVEIKRTVAAPSVFIFPPSDEQLK VYYCARWGYYGSGSYENDAFDIWGQGTMVT SGTASVVCLLNNFYPREAKVQWKVDNALQS VSSASTKGPSVFPLAPSSKSTSGGTAALGC GNSQESVTEQDSKDSTYSLSSTLTLSKADY LVKDYFPEPVTVSWNSGALTSGVHTFPAVL EKHKVYACEVTHQGLSSPVTKSFNRGEC QSSGLYSLSSVVTVPSSSLGTQTYICNVNH (SEQ ID NO: 53) KPSNTKVDKKVEPKCEF (SEQ ID NO: 54)

TABLE 5 PROTEASE-REGULATED ANTIBODIES (Type 2) IgG-like protease-regulated antibodies against TF and RG1 Light chain Heavy chain 3E10-Linkerl-19G9 NFMLTQPHSVSASPGKTVTISCTRSSGSVA QVNLRESGGTLVQPGGSLRLSCAASGFSFT SYYVQWYQQRPGSSPTTVIYEDNHRPSGVP DAWMSWVRQAPGKELEWVSSISGSGGSTYY DRFSGSIDTSSNSASLTISGLKTEDEADYY AGSVKGRFTISRDNSKNTLYLQMNSLRAED CQSYDSNNLVVFGGGTKLTVLGASDDDDKE TAVYYCARVLSLTDYYWYGMDVWGQGTLVT IVLTQSPGTLSLSPGERATLSCRASQSVSS VSASDDDDKEVQLVQSGGGLVQPGGSLRLS SYLAWYQQKPGQAPRLLIYGASSRATGIPD CAGSGFTFSSYVMHWLRQAPGKGLEWVSVI RFSGSGSGTDFTLTISRLEPEDFAVYYCQQ GTGGVTHYADSVKGRFTISRDNAKNSLYLQ YSSSLTFGGGTKVEIKRTVAAPSVFIFPPS MNSLRAEDTAVYYCARWGYYGSGSYENDAF DEQLKSGTASVVCLLNNFYPREAKVQWKVD DIWGQGTMVTVSSASTKGPSVFPLAPSSKS NALQSGNSQESVTEQDSKDSTYSLSSTLTL TSGGTAALGCLVKDYFPEPVTVSWNSGALT SKADYEKHKVYACEVTHQGLSSPVTKSFNR SGVHTFPAVLQSSGLYSLSSVVTVPSSSLG GEC TQTYICNVNHKPSNTKVDKRVEPKSCDKTH (SEQ ID NO: 55) TCPPCPAPELLGGPSVFLFPPKPKDTLMIS RTPEVTCVVVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRVVSVLTVLHQDWL NGKEYKCKVSNKALPAPIEKTISKAKGQPR EPQVYTLPPSREEMTKNQVSLTCLVKGFYP SDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSPGK (SEQ ID NO: 56) 3E10-Linker2-19G9 NFMLTQPHSVSASPGKTVTISCTRSSGSVA QVNLRESGGTLVQPGGSLRLSCAASGFSFT SYYVQWYQQRPGSSPTTVIYEDNHRPSGVP DAWMSWVRQAPGKELEWVSSISGSGGSTYY DRFSGSIDTSSNSASLTISGLKTEDEADYY AGSVKGRFTISRDNSKNTLYLQMNSLRAED CQSYDSNNLVVFGGGTKLTVLGAGGGGSDD TAVYYCARVLSLTDYYWYGMDVWGQGTLVT DDKEIVLTQSPGTLSLSPGERATLSCRASQ VSAGGGGSDDDDKEVQLVQSGGGLVQPGGS SVSSSYLAWYQQKPGQAPRLLIYGASSRAT LRLSCAGSGFTFSSYVMHWLRQAPGKGLEW GIPDRFSGSGSGTDFTLTISRLEPEDFAVY VSVIGTGGVTHYADSVKGRFTISRDNAKNS YCQQYSSSLTFGGGTKVEIKRTVAAPSVFI LYLQMNSLRAEDTAVYYCARWGYYGSGSYE FPPSDEQLKSGTASVVCLLNNFYPREAKVQ NDAFDIWGQGTMVTVSSASTKGPSVFPLAP WKVDNALQSGNSQESVTEQDSKDSTYSLSS SSKSTSGGTAALGCLVKDYFPEPVTVSWNS TLTLSKADYEKHKVYACEVTHQGLSSPVTK GALTSGVHTFPAVLQSSGLYSLSSVVTVPS SFNRGEC SSLGTQTYICNVNHKPSNTKVDKRVEPKSC (SEQ ID NO: 57) DKTHTCPPCPAPELLGGPSVFLFPPKPKDT LMISRTPEVTCVVVDVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQVYTLPPSREEMTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPGK (SEQ ID NO: 58) 3E10-Linker3-19G9 NFMLTQPHSVSASPGKTVTISCTRSSGSVA QVNLRESGGTLVQPGGSLRLSCAASGFSFT SYYVQWYQQRPGSSPTTVIYEDNHRPSGVP DAWMSWVRQAPGKELEWVSSISGSGGSTYY DRFSGSIDTSSNSASLTISGLKTEDEADYY AGSVKGRFTISRDNSKNTLYLQMNSLRAED CQSYDSNNLVVFGGGTKLTVLGAGGGGSDD TAVYYCARVLSLTDYYWYGMDVWGQGTLVT DDKGGGGSEIVLTQSPGTLSLSPGERATLS VSAGGGGSDDDDKGGGGSEVQLVQSGGGLV CRASQSVSSSYLAWYQQKPGQAPRLLIYGA QPGGSLRLSCAGSGFTFSSYVMHWLRQAPG SSRATGIPDRFSGSGSGTDFTLTISRLEPE KGLEWVSVIGTGGVTHYADSVKGRFTISRD DFAVYYCQQYSSSLTFGGGTKVEIKRTVAA NAKNSLYLQMNSLRAEDTAVYYCARWGYYG PSVFIFPPSDEQLKSGTASVVCLLNNFYPR SGSYENDAFDIWGQGTMVTVSSASTKGPSV EAKVQWKVDNALQSGNSQESVTEQDSKDST FPLAPSSKSTSGGTAALGCLVKDYFPEPVT YSLSSTLTLSKADYEKHKVYACEVTHQGLS VSWNSGALTSGVHTFPAVLQSSGLYSLSSV SPVTKSFNRGEC VTVPSSSLGTQTYICNVNHKPSNTKVDKRV (SEQ ID NO: 59) EPKSCDKTHTCPPCPAPELLGGPSVFLFPP KPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSREEMTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 60) 3E10-Linker4-19G9 NFMLTQPHSVSASPGKTVTISCTRSSGSVASYYVQ QVNLRESGGTLVQPGGSLRLSCAASGFSFTDAWMS WYQQRPGSSPTTVIYEDNHRPSGVPDRFSGSIDTS WVRQAPGKELEWVSSISGSGGSTYYAGSVKGRFTI SNSASLTISGLKTEDEADYYCQSYDSNNLVVFGGG SRDNSKNTLYLQMNSLRAEDTAVYYCARVLSLTDY TKLTVLGAGGGGSGGGGSGGGGSEIVLTQSPGTLS YWYGMDVWGQGTLVTVSAGGGGSGGGGSGGGGSEV LSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRL QLVQSGGGLVQPGGSLRLSCAGSGFTFSSYVMHWL LIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPE RQAPGKGLEWVSVIGTGGVTHYADSVKGRFTISRD DFAVYYCQQYSSSLTFGGGTKVEIKRTVAAPSVFI NAKNSLYLQMNSLRAEDTAVYYCARWGYYGSGSYE FPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDN NDAFDIWGQGTMVTVSSASTKGPSVFPLAPSSKST ALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYE SGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF KHKVYACEVTHQGLSSPVTKSFNRGEC PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHK (SEQ ID NO: 61) PSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSV FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPRE PQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 62) 3E10-Link1-19G9 Fab DIVLTQPHSVSASPGKTVTISCTRSSGSVASYYVQ QVQLVESGGTLVQPGGSLRLSCAASGFSFTDAWMS WYQQRPGSSPTTVIYEDNHRPSGVPDRFSGSIDTS WVRQAPGKELEWVSSISGSGGSTYYAGSVKGRFTI SNSASLTISGLKTEDEADYYCQSYDSNNLVVFGGG SRDNSKNTLYLQMNSLRAEDTAVYYCARVLSLTDY TKLTVLGASDDDDKEIVLTQSPGTLSLSPGERATL YWYGMDVWGQGTLVTVSASDDDDKEVQLVQSGGGL SCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRA VQPGGSLRLSCAGSGFTFSSYVMHWLRQAPGKGLE TGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQ WVSVIGTGGVTHYADSVKGRFTISRDNAKNSLYLQ YSSSLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLK MNSLRAEDTAVYYCARWGYYGSGSYENDAFDIWGQ SGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQE GTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGC SVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL THQGLSSPVTKSFNRGEA YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKK (SEQ ID NO: 63) VEPKSEF (SEQ ID NO: 64)

TABLE 6 PROTEASE-REGULATED ANTIBODIES (Type 3) Fab-like protease-regulated antibodies Light chain Heavy chain H1L5 DIVLTQPHSVSASPGKTVTISCTRSSGSVA QVQLVESGGTLVQPGGSLRLSCAASGFSFT SYYVQWYQQRPGSSPTTVIYEDNHRPSGVP DAWMSWVRQAPGKELEWVSSISGSGGSTYY DRFSGSIDTSSNSASLTISGLKTEDEADYY AGSVKGRFTISRDNSKNTLYLQMNSLRAED CQSYDSNNLVVFGGGTKLTVLGDDDDKLTQ TAVYYCARVLSLTDYYWYGMDVWGQGTLVT SPGTLSLSPGERATLSCRASQSVSSSYLAW VSASDDDDKEVQLVQSGGGLVQPGGSLRLS YQQKPGQAPRLLIYGASSRATGIPDRFSGS CAGSGFTFSSYVMHWLRQAPGKGLEWVSVI GSGTDFTLTISRLEPEDFAVYYCQQYSSSL GTGGVTHYADSVKGRFTISRDNAKNSLYLQ TFGGGTKVEIKRTVAAPSVFIFPPSDEQLK MNSLRAEDTAVYYCARWGYYGSGSYENDAF SGTASVVCLLNNFYPREAKVQWKVDNALQS DIWGQGTMVTVSSASTKGPSVFPLAPSSKS GNSQESVTEQDSKDSTYSLSSTLTLSKADY TSGGTAALGCLVKDYFPEPVTVSWNSGALT EKHKVYACEVTHQGLSSPVTKSFNRGEC SGVHTFPAVLQSSGLYSLSSVVTVPSSSLG (SEQ ID NO: 65) TQTYICNVNHKPSNTKVDKKVEPKCEF (SEQ ID NO: 66) H2L1 DIVLTQPHSVSASPGKTVTISCTRSSGSVA QVQLVESGGTLVQPGGSLRLSCAASGFSFT SYYVQWYQQRPGSSPTTVIYEDNHRPSGVP DAWMSWVRQAPGKELEWVSSISGSGGSTYY DRFSGSIDTSSNSASLTISGLKTEDEADYY AGSVKGRFTISRDNSKNTLYLQMNSLRAED CQSYDSNNLVVFGGGTKLTVLGASDDDDKE TAVYYCARVLSLTDYYWYGMDVWGQGTLVT IVLTQSPGTLSLSPGERATLSCRASQSVSS VDDDDKEVQLVQSGGGLVQPGGSLRLSCAG SYLAWYQQKPGQAPRLLIYGASSRATGIPD SGFTFSSYVMHWLRQAPGKGLEWVSVIGTG RFSGSGSGTDFTLTISRLEPEDFAVYYCQQ GVTHYADSVKGRFTISRDNAKNSLYLQMNS YSSSLTFGGGTKVEIKRTVAAPSVFIFPPS LRAEDTAVYYCARWGYYGSGSYENDAFDIW DEQLKSGTASVVCLLNNFYPREAKVQWKVD GQGTMVTVSSASTKGPSVFPLAPSSKSTSG NALQSGNSQESVTEQDSKDSTYSLSSTLTL GTAALGCLVKDYFPEPVTVSWNSGALTSGV SKADYEKHKVYACEVTHQGLSSPVTKSFNR HTFPAVLQSSGLYSLSSVVTVPSSSLGTQT GEC YICNVNHKPSNTKVDKKVEPKCEF (SEQ ID NO: 67) (SEQ ID NO: 68) H2L2 DIVLTQPHSVSASPGKTVTISCTRSSGSVA QVQLVESGGTLVQPGGSLRLSCAASGFSFT SYYVQWYQQRPGSSPTTVIYEDNHRPSGVP DAWMSWVRQAPGKELEWVSSISGSGGSTYY DRFSGSIDTSSNSASLTISGLKTEDEADYY AGSVKGRFTISRDNSKNTLYLQMNSLRAED CQSYDSNNLVVFGGGTKLTVLGDDDDKEIV TAVYYCARVLSLTDYYWYGMDVWGQGTLVT LTQSPGTLSLSPGERATLSCRASQSVSSSY VDDDDKEVQLVQSGGGLVQPGGSLRLSCAG LAWYQQKPGQAPRLLIYGASSRATGIPDRF SGFTFSSYVMHWLRQAPGKGLEWVSVIGTG SGSGSGTDFTLTISRLEPEDFAVYYCQQYS GVTHYADSVKGRFTISRDNAKNSLYLQMNS SSLTFGGGTKVEIKRTVAAPSVFIFPPSDE LRAEDTAVYYCARWGYYGSGSYENDAFDIW QLKSGTASVVCLLNNFYPREAKVQWKVDNA GQGTMVTVSSASTKGPSVFPLAPSSKSTSG LQSGNSQESVTEQDSKDSTYSLSSTLTLSK GTAALGCLVKDYFPEPVTVSWNSGALTSGV ADYEKHKVYACEVTHQGLSSPVTKSFNRGE HTFPAVLQSSGLYSLSSVVTVPSSSLGTQT C YICNVNHKPSNTKVDKKVEPKCEF (SEQ ID NO: 69) (SEQ ID NO: 70) H2L4 DIVLTQPHSVSASPGKTVTISCTRSSGSVA QVQLVESGGTLVQPGGSLRLSCAASGFSFT SYYVQWYQQRPGSSPTTVIYEDNHRPSGVP DAWMSWVRQAPGKELEWVSSISGSGGSTYY DRFSGSIDTSSNSASLTISGLKTEDEADYY AGSVKGRFTISRDNSKNTLYLQMNSLRAED CQSYDSNNLVVFGGGTKLTVLGASASDDDD TAVYYCARVLSLTDYYWYGMDVWGQGTLVT KLTQSPGTLSLSPGERATLSCRASQSVSSS VDDDDKEVQLVQSGGGLVQPGGSLRLSCAG YLAWYQQKPGQAPRLLIYGASSRATGIPDR SGFTFSSYVMHWLRQAPGKGLEWVSVIGTG FSGSGSGTDFTLTISRLEPEDFAVYYCQQY GVTHYADSVKGRFTISRDNAKNSLYLQMNS SSSLTFGGGTKVEIKRTVAAPSVFIFPPSD LRAEDTAVYYCARWGYYGSGSYENDAFDIW EQLKSGTASVVCLLNNFYPREAKVQWKVDN GQGTMVTVSSASTKGPSVFPLAPSSKSTSG ALQSGNSQESVTEQDSKDSTYSLSSTLTLS GTAALGCLVKDYFPEPVTVSWNSGALTSGV KADYEKHKVYACEVTHQGLSSPVTKSFNRG HTFPAVLQSSGLYSLSSVVTVPSSSLGTQT EC YICNVNHKPSNTKVDKKVEPKCEF (SEQ ID NO: 71) (SEQ ID NO: 72) H2L5 DIVLTQPHSVSASPGKTVTISCTRSSGSVA QVQLVESGGTLVQPGGSLRLSCAASGFSFT SYYVQWYQQRPGSSPTTVIYEDNHRPSGVP DAWMSWVRQAPGKELEWVSSISGSGGSTYY DRFSGSIDTSSNSASLTISGLKTEDEADYY AGSVKGRFTISRDNSKNTLYLQMNSLRAED CQSYDSNNLVVFGGGTKLTVLGDDDDKLTQ TAVYYCARVLSLTDYYWYGMDVWGQGTLVT SPGTLSLSPGERATLSCRASQSVSSSYLAW VDDDDKEVQLVQSGGGLVQPGGSLRLSCAG YQQKPGQAPRLLIYGASSRATGIPDRFSGS SGFTFSSYVMHWLRQAPGKGLEWVSVIGTG GSGTDFTLTISRLEPEDFAVYYCQQYSSSL GVTHYADSVKGRFTISRDNAKNSLYLQMNS TFGGGTKVEIKRTVAAPSVFIFPPSDEQLK LRAEDTAVYYCARWGYYGSGSYENDAFDIW SGTASVVCLLNNFYPREAKVQWKVDNALQS GQGTMVTVSSASTKGPSVFPLAPSSKSTSG GNSQESVTEQDSKDSTYSLSSTLTLSKADY GTAALGCLVKDYFPEPVTVSWNSGALTSGV EKHKVYACEVTHQGLSSPVTKSFNRGEC HTFPAVLQSSGLYSLSSVVTVPSSSLGTQT (SEQ ID NO: 73) YICNVNHKPSNTKVDKKVEPKCEF (SEQ ID NO: 74) H2L7 DIVLTQPHSVSASPGKTVTISCTRSSGSVA QVQLVESGGTLVQPGGSLRLSCAASGFSFT SYYVQWYQQRPGSSPTTVIYEDNHRPSGVP DAWMSWVRQAPGKELEWVSSISGSGGSTYY DRFSGSIDTSSNSASLTISGLKTEDEADYY AGSVKGRFTISRDNSKNTLYLQMNSLRAED CQSYDSNNLVVFGGGTKLTVLGASDDDDKS TAVYYCARVLSLTDYYWYGMDVWGQGTLVT PGTLSLSPGERATLSCRASQSVSSSYLAWY VDDDDKEVQLVQSGGGLVQPGGSLRLSCAG QQKPGQAPRLLIYGASSRATGIPDRFSGSG SGFTFSSYVMHWLRQAPGKGLEWVSVIGTG SGTDFTLTISRLEPEDFAVYYCQQYSSSLT GVTHYADSVKGRFTISRDNAKNSLYLQMNS FGGGTKVEIKRTVAAPSVFIFPPSDEQLKS LRAEDTAVYYCARWGYYGSGSYENDAFDIW GTASVVCLLNNFYPREAKVQWKVDNALQSG GQGTMVTVSSASTKGPSVFPLAPSSKSTSG NSQESVTEQDSKDSTYSLSSTLTLSKADYE GTAALGCLVKDYFPEPVTVSWNSGALTSGV KHKVYACEVTHQGLSSPVTKSFNRGEC HTFPAVLQSSGLYSLSSVVTVPSSSLGTQT (SEQ ID NO: 75) YICNVNHKPSNTKVDKKVEPKCEF (SEQ ID NO: 76) H2L8 DIVLTQPHSVSASPGKTVTISCTRSSGSVA QVQLVESGGTLVQPGGSLRLSCAASGFSFT SYYVQWYQQRPGSSPTTVIYEDNHRPSGVP DAWMSWVRQAPGKELEWVSSISGSGGSTYY DRFSGSIDTSSNSASLTISGLKTEDEADYY AGSVKGRFTISRDNSKNTLYLQMNSLRAED CQSYDSNNLVVFGGGTKLTVLGDDDDKSPG TAVYYCARVLSLTDYYWYGMDVWGQGTLVT TLSLSPGERATLSCRASQSVSSSYLAWYQQ VDDDDKEVQLVQSGGGLVQPGGSLRLSCAG KPGQAPRLLIYGASSRATGIPDRFSGSGSG SGFTFSSYVMHWLRQAPGKGLEWVSVIGTG TDFTLTISRLEPEDFAVYYCQQYSSSLTFG GVTHYADSVKGRFTISRDNAKNSLYLQMNS GGTKVEIKRTVAAPSVFIFPPSDEQLKSGT LRAEDTAVYYCARWGYYGSGSYENDAFDIW ASVVCLLNNFYPREAKVQWKVDNALQSGNS GQGTMVTVSSASTKGPSVFPLAPSSKSTSG QESVTEQDSKDSTYSLSSTLTLSKADYEKH GTAALGCLVKDYFPEPVTVSWNSGALTSGV KVYACEVTHQGLSSPVTKSFNRGEC HTFPAVLQSSGLYSLSSVVTVPSSSLGTQT (SEQ ID NO: 77) YICNVNHKPSNTKVDKKVEPKCEF (SEQ ID NO: 78)

TABLE 7 PROTEASE-REGULATED ANTIBODIES (Type 3) IgG-like protease-regulated antibodies Light chain Heavy chain 3E10-Linkerl a-19G9 NFMLTQPHSVSASPGKTVTISCTRSSGSVA QVNLRESGGTLVQPGGSLRLSCAASGFSFT SYYVQWYQQRPGSSPTTVIYEDNHRPSGVP DAWMSWVRQAPGKELEWVSSISGSGGSTYY DRFSGSIDTSSNSASLTISGLKTEDEADYY AGSVKGRFTISRDNSKNTLYLQMNSLRAED CQSYDSNNLVVFGGGTKLTVSDDDDKEIVL TAVYYCARVLSLTDYYWYGMDVWGQGTLVT TQSPGTLSLSPGERATLSCRASQSVSSSYL VDDDDKEVQLVQSGGGLVQPGGSLRLSCAG AWYQQKPGQAPRLLIYGASSRATGIPDRFS SGFTFSSYVMHWLRQAPGKGLEWVSVIGTG GSGSGTDFTLTISRLEPEDFAVYYCQQYSS GVTHYADSVKGRFTISRDNAKNSLYLQMNS SLTFGGGTKVEIKRTVAAPSVFIFPPSDEQ LRAEDTAVYYCARWGYYGSGSYENDAFDIW LKSGTASVVCLLNNFYPREAKVQWKVDNAL GQGTMVTVSSASTKGPSVFPLAPSSKSTSG QSGNSQESVTEQDSKDSTYSLSSTLTLSKA GTAALGCLVKDYFPEPVTVSWNSGALTSGV DYEKHKVYACEVTHQGLSSPVTKSFNRGEC HTFPAVLQSSGLYSLSSVVTVPSSSLGTQT (SEQ ID NO: 79) YICNVNHKPSNTKVDKRVEPKSCDKTHTCP PCPAPELLGGPSVFLFPPKPKDTLMISRTP EVTCVVVDVSHEDPEVKFNWYVDGVEVHNA KTKPREEQYNSTYRVVSVLTVLHQDWLNGK EYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSREEMTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGK (SEQ ID NO: 80) 3E10-Linkerlb-19G9 NFMLTQPHSVSASPGKTVTISCTRSSGSVA QVNLRESGGTLVQPGGSLRLSCAASGFSFT SYYVQWYQQRPGSSPTTVIYEDNHRPSGVP DAWMSWVRQAPGKELEWVSSISGSGGSTYY DRFSGSIDTSSNSASLTISGLKTEDEADYY AGSVKGRFTISRDNSKNTLYLQMNSLRAED CQSYDSNNLVVFGGGTKLTVSDDDDKLTQS TAVYYCARVLSLTDYYWYGMDVWGQGTLVT PGTLSLSPGERATLSCRASQSVSSSYLAWY VDDDDKQSGGGLVQPGGSLRLSCAGSGFTF QQKPGQAPRLLIYGASSRATGIPDRFSGSG SSYVMHWLRQAPGKGLEWVSVIGTGGVTHY SGTDFTLTISRLEPEDFAVYYCQQYSSSLT ADSVKGRFTISRDNAKNSLYLQMNSLRAED FGGGTKVEIKRTVAAPSVFIFPPSDEQLKS TAVYYCARWGYYGSGSYENDAFDIWGQGTM GTASVVCLLNNFYPREAKVQWKVDNALQSG VTVSSASTKGPSVFFLAPSSKSTSGGTAAL NSQESVTEQDSKDSTYSLSSTLTLSKADYE GCLVKDYFPEPVTVSWNSGALTSGVHTFPA KHKVYACEVTHQGLSSPVTKSFNRGEC VLQSSGLYSLSSVVTVPSSSLGTQTYICNV (SEQ ID NO: 81) NHKPSNTKVDKRVEPKSCDKTHTCPPCPAP ELLGGPSVFLFPPKPKDTLMISRTPEVTCV VVDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQYNSTYRVVSVLTVLHQDWLNGKEYKCK VSNKALPAPIEKTISKAKGQPREPQVYTLP PSREEMTKNQVSLTCLVKGFYPSDIAVEWE SNGQPENNYKTTPPVLDSDGSFFLYSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGK (SEQ ID NO: 82) 3E10-Linkerlc-19G9 NFMLTQPHSVSASPGKTVTISCTRSSGSVA QVNLRESGGTLVQPGGSLRLSCAASGFSFT SYYVQWYQQRPGSSPTTVIYEDNHRPSGVP DAWMSWVRQAPGKELEWVSSISGSGGSTYY DRFSGSIDTSSNSASLTISGLKTEDEADYY AGSVKGRFTISRDNSKNTLYLQMNSLRAED CQSYDSNNLVVFGGGTKLTVLGASDDDDKL TAVYYCARVLSLTDYYWYGMDVWGQGTLVT TQSPGTLSLSPGERATLSCRASQSVSSSYL VSASDDDDKQSGGGLVQPGGSLRLSCAGSG AWYQQKPGQAPRLLIYGASSRATGIPDRFS FTFSSYVMHWLRQAPGKGLEWVSVIGTGGV GSGSGTDFTLTISRLEPEDFAVYYCQQYSS THYADSVKGRFTISRDNAKNSLYLQMNSLR SLTFGGGTKVEIKRTVAAPSVFIFPPSDEQ AEDTAVYYCARWGYYGSGSYENDAFDIWGQ LKSGTASVVCLLNNFYPREAKVQWKVDNAL GTMVTVSSASTKGPSVFPLAPSSKSTSGGT QSGNSQESVTEQDSKDSTYSLSSTLTLSKA AALGCLVKDYFPEPVTVSWNSGALTSGVHT DYEKHKVYACEVTHQGLSSPVTKSFNRGEC FPAVLQSSGLYSLSSVVTVPSSSLGTQTYI (SEQ ID NO: 83) CNVNHKPSNTKVDKRVEPKSCDKTHTCPPC PAPELLGGPSVFLFPPKPKDTLMISRTPEV TCVVVDVSHEDPEVKFNWYVDGVEVHNAKT KPREEQYNSTYRVVSVLTVLHQDWLNGKEY KCKVSNKALPAPIEKTISKAKGQPREPQVY TLPPSREEMTKNQVSLTCLVKGFYPSDIAV EWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK (SEQ ID NO: 84)

TABLE 8 PROTEASE-REGULATED ANTIBODIES (Type 4) Light chain Heavy chain H3L1 DIVLTQPHSVSASPGKTVTISCTRSSGSVA QVQLVESGGTLVQPGGSLRLSCAASGFSFT SYYVQWYQQRPGSSPTTVIYEDNHRPSGVP DAWMSWVRQAPGKELEWVSSISGSGGSTYY DRFSGSIDTSSNSASLTISGLKTEDEADYY AGSVKGRFTISRDNSKNTLYLQMNSLRAED CQSYDSNNLVVFGGGTKLTVLGASDDDDKE TAVYYCARVLSLTDYYWYGMDVWGQGTLVT IVLTQSPGTLSLSPGERATLSCRASQSVSS VSASDDDDKQSGGGLVQPGGSLRLSCAGSG SYLAWYQQKPGQAPRLLIYGASSRATGIPD FTFSSYVMHWLRQAPGKGLEWVSVIGTGGV RFSGSGSGTDFTLTISRLEPEDFAVYYCQQ THYADSVKGRFTISRDNAKNSLYLQMNSLR YSSSLTFGGGTKVEIKRTVAAPSVFIFPPS AEDTAVYYCARWGYYGSGSYENDAFDIWGQ DEQLKSGTASVVCLLNNFYPREAKVQWKVD GTMVTVSSASTKGPSVFPLAPSSKSTSGGT NALQSGNSQESVTEQDSKDSTYSLSSTLTL AALGCLVKDYFPEPVTVSWNSGALTSGVHT SKADYEKHKVYACEVTHQGLSSPVTKSFNR FPAVLQSSGLYSLSSVVTVPSSSLGTQTYI GEC CNVNHKPSNTKVDKKVEPKCEF (SEQ ID NO: 85) (SEQ ID NO: 86) H3L2 DIVLTQPHSVSASPGKTVTISCTRSSGSVA QVQLVESGGTLVQPGGSLRLSCAASGFSFT SYYVQWYQQRPGSSPTTVIYEDNHRPSGVP DAWMSWVRQAPGKELEWVSSISGSGGSTYY DRFSGSIDTSSNSASLTISGLKTEDEADYY AGSVKGRFTISRDNSKNTLYLQMNSLRAED CQSYDSNNLVVFGGGTKLTVLGDDDDKEIV TAVYYCARVLSLTDYYWYGMDVWGQGTLVT LTQSPGTLSLSPGERATLSCRASQSVSSSY VSASDDDDKQSGGGLVQPGGSLRLSCAGSG LAWYQQKPGQAPRLLIYGASSRATGIPDRF FTFSSYVMHWLRQAPGKGLEWVSVIGTGGV SGSGSGTDFTLTISRLEPEDFAVYYCQQYS THYADSVKGRFTISRDNAKNSLYLQMNSLR SSLTFGGGTKVEIKRTVAAPSVFIFPPSDE AEDTAVYYCARWGYYGSGSYENDAFDIWGQ QLKSGTASVVCLLNNFYPREAKVQWKVDNA GTMVTVSSASTKGPSVFPLAPSSKSTSGGT LQSGNSQESVTEQDSKDSTYSLSSTLTLSK AALGCLVKDYFPEPVTVSWNSGALTSGVHT ADYEKHKVYACEVTHQGLSSPVTKSFNRGE FPAVLQSSGLYSLSSVVTVPSSSLGTQTYI C CNVNHKPSNTKVDKKVEPKCEF (SEQ ID NO: 87) (SEQ ID NO: 88) H3L4 DIVLTQPHSVSASPGKTVTISCTRSSGSVA QVQLVESGGTLVQPGGSLRLSCAASGFSFT SYYVQWYQQRPGSSPTTVIYEDNHRPSGVP DAWMSWVRQAPGKELEWVSSISGSGGSTYY DRFSGSIDTSSNSASLTISGLKTEDEADYY AGSVKGRFTISRDNSKNTLYLQMNSLRAED CQSYDSNNLVVFGGGTKLTVLGASDDDDKL TAVYYCARVLSLTDYYWYGMDVWGQGTLVT TQSPGTLSLSPGERATLSCRASQSVSSSYL VSASDDDDKQSGGGLVQPGGSLRLSCAGSG AWYQQKPGQAPRLLIYGASSRATGIPDRFS FTFSSYVMHWLRQAPGKGLEWVSVIGTGGV GSGSGTDFTLTISRLEPEDFAVYYCQQYSS THYADSVKGRFTISRDNAKNSLYLQMNSLR SLTFGGGTKVEIKRTVAAPSVFIFPPSDEQ AEDTAVYYCARWGYYGSGSYENDAFDIWGQ LKSGTASVVCLLNNFYPREAKVQWKVDNAL GTMVTVSSASTKGPSVFPLAPSSKSTSGGT QSGNSQESVTEQDSKDSTYSLSSTLTLSKA AALGCLVKDYFPEPVTVSWNSGALTSGVHT DYEKHKVYACEVTHQGLSSPVTKSFNRGEC FPAVLQSSGLYSLSSVVTVPSSSLGTQTYI (SEQ ID NO: 89) CNVNHKPSNTKVDKKVEPKCEF (SEQ ID NO: 90) H3L5 DIVLTQPHSVSASPGKTVTISCTRSSGSVA QVQLVESGGTLVQPGGSLRLSCAASGFSFT SYYVQWYQQRPGSSPTTVIYEDNHRPSGVP DAWMSWVRQAPGKELEWVSSISGSGGSTYY DRFSGSIDTSSNSASLTISGLKTEDEADYY AGSVKGRFTISRDNSKNTLYLQMNSLRAED CQSYDSNNLVVFGGGTKLTVLGDDDDKLTQ TAVYYCARVLSLTDYYWYGMDVWGQGTLVT SPGTLSLSPGERATLSCRASQSVSSSYLAW VSASDDDDKQSGGGLVQPGGSLRLSCAGSG YQQKPGQAPRLLIYGASSRATGIPDRFSGS FTFSSYVMHWLRQAPGKGLEWVSVIGTGGV GSGTDFTLTISRLEPEDFAVYYCQQYSSSL THYADSVKGRFTISRDNAKNSLYLQMNSLR TFGGGTKVEIKRTVAAPSVFIFPPSDEQLK AEDTAVYYCARWGYYGSGSYENDAFDIWGQ SGTASVVCLLNNFYPREAKVQWKVDNALQS GTMVTVSSASTKGPSVFPLAPSSKSTSGGT GNSQESVTEQDSKDSTYSLSSTLTLSKADY AALGCLVKDYFPEPVTVSWNSGALTSGVHT EKHKVYACEVTHQGLSSPVTKSFNRGEC FPAVLQSSGLYSLSSVVTVPSSSLGTQTYI (SEQ ID NO: 91) CNVNHKPSNTKVDKKVEPKCEF (SEQ ID NO: 92) H3L7 DIVLTQPHSVSASPGKTVTISCTRSSGSVA QVQLVESGGTLVQPGGSLRLSCAASGFSFT SYYVQWYQQRPGSSPTTVIYEDNHRPSGVP DAWMSWVRQAPGKELEWVSSISGSGGSTYY DRFSGSIDTSSNSASLTISGLKTEDEADYY AGSVKGRFTISRDNSKNTLYLQMNSLRAED CQSYDSNNLVVFGGGTKLTVLGASDDDDKS TAVYYCARVLSLTDYYWYGMDVWGQGTLVT PGTLSLSPGERATLSCRASQSVSSSYLAWY VSASDDDDKQSGGGLVQPGGSLRLSCAGSG QQKPGQAPRLLIYGASSRATGIPDRFSGSG FTFSSYVMHWLRQAPGKGLEWVSVIGTGGV SGTDFTLTISRLEPEDFAVYYCQQYSSSLT THYADSVKGRFTISRDNAKNSLYLQMNSLR FGGGTKVEIKRTVAAPSVFIFPPSDEQLKS AEDTAVYYCARWGYYGSGSYENDAFDIWGQ GTASVVCLLNNFYPREAKVQWKVDNALQSG GTMVTVSSASTKGPSVFPLAPSSKSTSGGT NSQESVTEQDSKDSTYSLSSTLTLSKADYE AALGCLVKDYFPEPVTVSWNSGALTSGVHT KHKVYACEVTHQGLSSPVTKSFNRGEC FPAVLQSSGLYSLSSVVTVPSSSLGTQTYI (SEQ ID NO: 93) CNVNHKPSNTKVDKKVEPKCEF (SEQ ID NO: 94) H1L2 DIVLTQPHSVSASPGKTVTISCTRSSGSVA QVQLVESGGTLVQPGGSLRLSCAASGFSFT SYYVQWYQQRPGSSPTTVIYEDNHRPSGVP DAWMSWVRQAPGKELEWVSSISGSGGSTYY DRFSGSIDTSSNSASLTISGLKTEDEADYY AGSVKGRFTISRDNSKNTLYLQMNSLRAED CQSYDSNNLVVFGGGTKLTVLGDDDDKEIV TAVYYCARVLSLTDYYWYGMDVWGQGTLVT LTQSPGTLSLSPGERATLSCRASQSVSSSY VSASDDDDKEVQLVQSGGGLVQPGGSLRLS LAWYQQKPGQAPRLLIYGASSRATGIPDRF CAGSGFTFSSYVMHWLRQAPGKGLEWVSVI SGSGSGTDFTLTISRLEPEDFAVYYCQQYS GTGGVTHYADSVKGRFTISRDNAKNSLYLQ SSLTFGGGTKVEIKRTVAAPSVFIFPPSDE MNSLRAEDTAVYYCARWGYYGSGSYENDAF QLKSGTASVVCLLNNFYPREAKVQWKVDNA DIWGQGTMVTVSSASTKGPSVFPLAPSSKS LQSGNSQESVTEQDSKDSTYSLSSTLTLSK TSGGTAALGCLVKDYFPEPVTVSWNSGALT ADYEKHKVYACEVTHQGLSSPVTKSFNRGE SGVHTFPAVLQSSGLYSLSSVVTVPSSSLG C TQTYICNVNHKPSNTKVDKKVEPKCEF (SEQ ID NO: 95) (SEQ ID NO: 96) H5L1 DIVLTQPHSVSASPGKTVTISCTRSSGSVA QVQLVESGGTLVQPGGSLRLSCAASGFSFT SYYVQWYQQRPGSSPTTVIYEDNHRPSGVP DAWMSWVRQAPGKELEWVSSISGSGGSTYY DRFSGSIDTSSNSASLTISGLKTEDEADYY AGSVKGRFTISRDNSKNTLYLQMNSLRAED CQSYDSNNLVVFGGGTKLTVLGASDDDDKE TAVYYCARVLSLTDYYWYGMDVWGQGTLVT IVLTQSPGTLSLSPGERATLSCRASQSVSS VSASDDDDKLVQPGGSLRLSCAGSGFTFSS SYLAWYQQKPGQAPRLLIYGASSRATGIPD YVMHWLRQAPGKGLEWVSVIGTGGVTHYAD RFSGSGSGTDFTLTISRLEPEDFAVYYCQQ SVKGRFTISRDNAKNSLYLQMNSLRAEDTA YSSSLTFGGGTKVEIKRTVAAPSVFIFPPS VYYCARWGYYGSGSYENDAFDIWGQGTMVT DEQLKSGTASVVCLLNNFYPREAKVQWKVD VSSASTKGPSVFPLAPSSKSTSGGTAALGC NALQSGNSQESVTEQDSKDSTYSLSSTLTL LVKDYFPEPVTVSWNSGALTSGVHTFPAVL SKADYEKHKVYACEVTHQGLSSPVTKSFNR QSSGLYSLSSVVTVPSSSLGTQTYICNVNH GEC KPSNTKVDKKVEPKCEF (SEQ ID NO: 97) (SEQ ID NO: 98) H5L4 DIVLTQPHSVSASPGKTVTISCTRSSGSVA QVQLVESGGTLVQPGGSLRLSCAASGFSFT SYYVQWYQQRPGSSPTTVIYEDNHRPSGVP DAWMSWVRQAPGKELEWVSSISGSGGSTYY DRFSGSIDTSSNSASLTISGLKTEDEADYY AGSVKGRFTISRDNSKNTLYLQMNSLRAED CQSYDSNNLVVFGGGTKLTVLGASDDDDKL TAVYYCARVLSLTDYYWYGMDVWGQGTLVT TQSPGTLSLSPGERATLSCRASQSVSSSYL VSASDDDDKLVQPGGSLRLSCAGSGFTFSS AWYQQKPGQAPRLLIYGASSRATGIPDRFS YVMHWLRQAPGKGLEWVSVIGTGGVTHYAD GSGSGTDFTLTISRLEPEDFAVYYCQQYSS SVKGRFTISRDNAKNSLYLQMNSLRAEDTA SLTEGGGTKVEIKRTVAAPSVFIFPPSDEQ VYYCARWGYYGSGSYENDAFDIWGQGTMVT LKSGTASVVCLLNNFYPREAKVQWKVDNAL VSSASTKGPSVFPLAPSSKSTSGGTAALGC QSGNSQESVTEQDSKDSTYSLSSTLTLSKA LVKDYFPEPVTVSWNSGALTSGVHTFPAVL DYEKHKVYACEVTHQGLSSPVTKSFNRGEC QSSGLYSLSSVVTVPSSSLGTQTYICNVNH (SEQ ID NO: 99) KPSNTKVDKKVEPKCEF (SEQ ID NO: 100) H5L7 DIVLTQPHSVSASPGKTVTISCTRSSGSVA QVQLVESGGTLVQPGGSLRLSCAASGFSFT SYYVQWYQQRPGSSPTTVIYEDNHRPSGVP DAWMSWVRQAPGKELEWVSSISGSGGSTYY DRFSGSIDTSSNSASLTISGLKTEDEADYY AGSVKGRFTISRDNSKNTLYLQMNSLRAED CQSYDSNNLVVFGGGTKLTVLGASDDDDKS TAVYYCARVLSLTDYYWYGMDVWGQGTLVT PGTLSLSPGERATLSCRASQSVSSSYLAWY VSASDDDDKLVQPGGSLRLSCAGSGFTFSS QQKPGQAPRLLIYGASSRATGIPDRFSGSG YVMHWLRQAPGKGLEWVSVIGTGGVTHYAD SGTDFTLTISRLEPEDFAVYYCQQYSSSLT SVKGRFTISRDNAKNSLYLQMNSLRAEDTA FGGGTKVEIKRTVAAPSVFIFPPSDEQLKS VYYCARWGYYGSGSYENDAFDIWGQGTMVT GTASVVCLLNNFYPREAKVQWKVDNALQSG VSSASTKGPSVFPLAPSSKSTSGGTAALGC NSQESVTEQDSKDSTYSLSSTLTLSKADYE LVKDYFPEPVTVSWNSGALTSGVHTFPAVL KHKVYACEVTHQGLSSPVTKSFNRGEC QSSGLYSLSSVVTVPSSSLGTQTYICNVNH (SEQ ID NO: 101) KPSNTKVDKKVEPKCEF (SEQ ID NO: 102) H5L8 DIVLTQPHSVSASPGKTVTISCTRSSGSVA QVQLVESGGTLVQPGGSLRLSCAASGFSFT SYYVQWYQQRPGSSPTTVIYEDNHRPSGVP DAWMSWVRQAPGKELEWVSSISGSGGSTYY DRFSGSIDTSSNSASLTISGLKTEDEADYY AGSVKGRFTISRDNSKNTLYLQMNSLRAED CQSYDSNNLVVFGGGTKLTVLGDDDDKSPG TAVYYCARVLSLTDYYWYGMDVWGQGTLVT TLSLSPGERATLSCRASQSVSSSYLAWYQQ VSASDDDDKLVQPGGSLRLSCAGSGFTFSS KPGQAPRLLIYGASSRATGIPDRFSGSGSG YVMHWLRQAPGKGLEWVSVIGTGGVTHYAD TDFTLTISRLEPEDFAVYYCQQYSSSLTFG SVKGRFTISRDNAKNSLYLQMNSLRAEDTA GGTKVEIKRTVAAPSVFIFPPSDEQLKSGT VYYCARWGYYGSGSYENDAFDIWGQGTMVT ASVVCLLNNFYPREAKVQWKVDNALQSGNS VSSASTKGPSVFPLAPSSKSTSGGTAALGC QESVTEQDSKDSTYSLSSTLTLSKADYEKH LVKDYFPEPVTVSWNSGALTSGVHTFPAVL KVYACEVTHQGLSSPVTKSFNRGEC QSSGLYSLSSVVTVPSSSLGTQTYICNVNH (SEQ ID NO: 103) KPSNTKVDKKVEPKCEF (SEQ ID NO: 104) H6L1 DIVLTQPHSVSASPGKTVTISCTRSSGSVA QVQLVESGGTLVQPGGSLRLSCAASGFSFT SYYVQWYQQRPGSSPTTVIYEDNHRPSGVP DAWMSWVRQAPGKELEWVSSISGSGGSTYY DRFSGSIDTSSNSASLTISGLKTEDEADYY AGSVKGRFTISRDNSKNTLYLQMNSLRAED CQSYDSNNLVVFGGGTKLTVLGASDDDDKE TAVYYCARVLSLTDYYWYGMDVWGQGTLVT IVLTQSPGTLSLSPGERATLSCRASQSVSS VDDDDKLVQPGGSLRLSCAGSGFTFSSYVM SYLAWYQQKPGQAPRLLIYGASSRATGIPD HWLRQAPGKGLEWVSVIGTGGVTHYADSVK RFSGSGSGTDFTLTISRLEPEDFAVYYCQQ GRFTISRDNAKNSLYLQNNSLRAEDTAVYY YSSSLTFGGGTKVEIKRTVAAPSVFIFPPS CARWGYYGSGSYENDAFDIWGQGTMVTVSS DEQLKSGTASVVCLLNNFYPREAKVQWKVD ASTKGPSVFPLAPSSKSTSGGTAALGCLVK NALQSGNSQESVTEQDSKDSTYSLSSTLTL DYFPEPVTVSWNSGALTSGVHTFPAVLQSS SKADYEKHKVYACEVTHQGLSSPVTKSFNR GLYSLSSVVTVPSSSLGTQTYICNVNHKPS GEC NTKVDKKVEPKCEF (SEQ ID NO: 105) (SEQ ID NO: 106)

TABLE 9 PROTEASE-REGULATED ANTIBODIES (Type 4) Heavy chain Heavy chain H1L5a QVQLVESGGTLVQPGGSLRLSCAASGFSFT QVQLVESGGTLVQPGGSLRLSCAASGFSFT DAWMSWVRQAPGKELEWVSSISGSGGSTYY DAWMSWVRQAPGKELEWVSSISGSGGSTYY AGSVKGRFTISRDNSKNTLYLQMNSLRAED AGSVKGRFTISRDNSKNTLYLQMNSLRAED TAVYYCARAAAAAAAAAAAAAAWGQGTLVT TAVYYCARAAAAAAAAAAAAAAWGQGTLVT VSASDDDDKEVQLVQSGGGLVQPGGSLRLS VSASDDDDKEVQLVQSGGGLVQPGGSLRLS CAGSGFTFSSYVMHWLRQAPGKGLEWVSVI CAGSGFTFSSYVMHWLRQAPGKGLEWVSVI GTGGVTHYADSVKGRFTISRDNAKNSLYLQ GTGGVTHYADSVKGRFTISRDNAKNSLYLQ MNSLRAEDTAVYYCARWGYYGSGSYENDAF MNSLRAEDTAVYYCARWGYYGSGSYENDAF DIWGQGTMVTVSSASTKGPSVFPLAPSSKS DIWGQGTMVTVSSASTKGPSVFPLAPSSKS TSGGTAALGCLVKDYFPEPVTVSWNSGALT TSGGTAALGCLVKDYFPEPVTVSWNSGALT SGVHTFPAVLQSSGLYSLSSVVTVPSSSLG SGVHTFPAVLQSSGLYSLSSVVTVPSSSLG TQTYICNVNHKPSNTKVDKKVEPKCEF TQTYICNVMHKPSNTICVDKICVEPKCEF (SEQ ID NO: 107) (SEQ ID NO: 108) H2L1a QVQLVESGGTLVQPGGSLRLSCAASGFSFT QVQLVESGGTLVQPGGSLRLSCAASGFSFT DAWMSWVRQAPGKELEWVSSISGSGGSTYY DAWMSWVRQAPGKELEWVSSISGSGGSTYY AGSVKGRFTISRDNSKNTLYLQMNSLRAED AGSVKGRFTISRDNSKNTLYLQMNSLRAED TAVYYCARAAAAAAAAAAAAAAWGQGTLVT TAVYYCARAAAAAAAAAAAAAAWGQGTLVT VDDDDKEVQLVQSGGGLVQPGGSLRLSCAG VDDDDKEVQLVQSGGGLVQPGGSLRLSCAG SGFTFSSYVMHWLRQAPGKGLEWVSVIGTG SGFTFSSYVMHWLRQAPGKGLEWVSVIGTG GVTHYADSVKGRFTISRDNAKNSLYLQMNS GVTHYADSVKGRFTISRDNAKNSLYLQMNS LRAEDTAVYYCARWGYYGSGSYENDAFDIW LRAEDTAVYYCARWGYYGSGSYENDAFDIW GQGTMVTVSSASTKGPSVFPLAPSSKSTSG GQGTMVTVSSASTKGPSVFPLAPSSKSTSG GTAALGCLVKDYFPEPVTVSWNSGALTSGV GTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSVVTVPSSSLGTQT HTFPAVLQSSGLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKKVEPKCEF YICNVNHKPSNTKVDKKVEPKCEF (SEQ ID NO: 109) (SEQ ID NO: 110) H2L2a QVQLVESGGTLVQPGGSLRLSCAASGFSFT QVQLVESGGTLVQPGGSLRLSCAASGFSFT DAWMSWVRQAPGKELEWVSSISGSGGSTYY DAWMSWVRQAPGKELEWVSSISGSGGSTYY AGSVKGRFTISRDNSKNTLYLQMNSLRAED AGSVKGRFTISRDNSKNTLYLQMNSLRAED TAVYYCARAAAAAAAAAAAAAAWGQGTLVT TAVYYCARAAAAAAAAAAAAAAWGQGTLVT VDDDDKEVQLVQSGGGLVQPGGSLRLSCAG VDDDDKEVQLVQSGGGLVQPGGSLRLSCAG SGFTFSSYVMHWLRQAPGKGLEWVSVIGTG SGFTFSSYVMHWLRQAPGKGLEWVSVIGTG GVTHYADSVKGRFTISRDNAKNSLYLQMNS GVTHYADSVKGRFTISRDNAKNSLYLQMNS LRAEDTAVYYCARWGYYGSGSYENDAFDIW LRAEDTAVYYCARWGYYGSGSYENDAFDIW GQGTMVTVSSASTKGPSVFPLAPSSKSTSG GQGTMVTVSSASTKGPSVFPLAPSSKSTSG GTAALGCLVKDYFPEPVTVSWNSGALTSGV GTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSVVTVPSSSLGTQT HTFPAVLQSSGLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKKVEPKCEF YICNVNHKPSNTKVDKKVEPKCEF (SEQ ID NO: 111) (SEQ ID NO: 112) H2L4a QVQLVESGGTLVQPGGSLRLSCAASGFSFT QVQLVESGGTLVQPGGSLRLSCAASGFSFT DAWMSWVRQAPGKELEWVSSISGSGGSTYY DAWMSWVRQAPGKELEWVSSISGSGGSTYY AGSVKGRFTISRDNSKNTLYLQMNSLRAED AGSVKGRFTISRDNSKNTLYLQMNSLRAED TAVYYCARAAAAAAAAAAAAAAWGQGTLVT TAVYYCARAAAAAAAAAAAAAAWGQGTLVT VDDDDKEVQLVQSGGGLVQPGGSLRLSCAG VDDDDKEVQLVQSGGGLVQPGGSLRLSCAG SGFTFSSYVMHWLRQAPGKGLEWVSVIGTG SGFTFSSYVMHWLRQAPGKGLEWVSVIGTG GVTHYADSVKGRFTISRDNAKNSLYLQMNS GVTHYADSVKGRFTISRDNAKNSLYLQMNS LRAEDTAVYYCARWGYYGSGSYENDAFDIW LRAEDTAVYYCARWGYYGSGSYENDAFDIW GQGTMVTVSSASTKGPSVFPLAPSSKSTSG GQGTMVTVSSASTKGPSVFPLAPSSKSTSG GTAALGCLVKDYFPEPVTVSWNSGALTSGV GTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSVVTVPSSSLGTQT HTFPAVLQSSGLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKKVEPKCEF YICNVNHKPSNTICVDKICVEPKCEF (SEQ ID NO: 113) (SEQ ID NO: 114) H2L5a QVQLVESGGTLVQPGGSLRLSCAASGFSFT QVQLVESGGTLVQPGGSLRLSCAASGFSFT DAWMSWVRQAPGKELEWVSSISGSGGSTYY DAWMSWVRQAPGKELEWVSSISGSGGSTYY AGSVKGRFTISRDNSKNTLYLQMNSLRAED AGSVKGRFTISRDNSKNTLYLQMNSLRAED TAVYYCARAAAAAAAAAAAAAAWGQGTLVT TAVYYCARAAAAAAAAAAAAAAWGQGTLVT VDDDDKEVQLVQSGGGLVQPGGSLRLSCAG VDDDDKEVQLVQSGGGLVQPGGSLRLSCAG SGFTFSSYVMHWLRQAPGKGLEWVSVIGTG SGFTFSSYVMHWLRQAPGKGLEWVSVIGTG GVTHYADSVKGRFTISRDNAKNSLYLQMNS GVTHYADSVKGRFTISRDNAKNSLYLQMNS LRAEDTAVYYCARWGYYGSGSYENDAFDIW LRAEDTAVYYCARWGYYGSGSYENDAFDIW GQGTMVTVSSASTKGPSVFPLAPSSKSTSG GQGTMVTVSSASTKGPSVFPLAPSSKSTSG GTAALGCLVKDYFPEPVTVSWNSGALTSGV GTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSVVTVPSSSLGTQT HTFPAVLQSSGLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKKVEPKCEF YICNVNHKPSNTKVDKKVEPKCEF (SEQ ID NO: 115) (SEQ ID NO: 116) H2L7a QVQLVESGGTLVQPGGSLRLSCAASGFSFT QVQLVESGGTLVQPGGSLRLSCAASGFSFT DAWMSWVRQAPGKELEWVSSISGSGGSTYY DAWMSWVRQAPGKELEWVSSISGSGGSTYY AGSVKGRFTISRDNSKNTLYLQMNSLRAED AGSVKGRFTISRDNSKNTLYLQMNSLRAED TAVYYCARAAAAAAAAAAAAAAWGQGTLVT TAVYYCARAAAAAAAAAAAAAAWGQGTLVT VDDDDKEVQLVQSGGGLVQPGGSLRLSCAG VDDDDKEVQLVQSGGGLVQPGGSLRLSCAG SGFTFSSYVMHWLRQAPGKGLEWVSVIGTG SGFTFSSYVMHWLRQAPGKGLEWVSVIGTG GVTHYADSVKGRFTISRDNAKNSLYLQMNS GVTHYADSVKGRFTISRDNAKNSLYLQMNS LRAEDTAVYYCARWGYYGSGSYENDAFDIW LRAEDTAVYYCARWGYYGSGSYENDAFDIW GQGTMVTVSSASTKGPSVFPLAPSSKSTSG GQGTMVTVSSASTKGPSVFPLAPSSKSTSG GTAALGCLVKDYFPEPVTVSWNSGALTSGV GTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSVVTVPSSSLGTQT HTFPAVLQSSGLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKKVEPKCEF YICNVNHKPSNTKVDKKVEPKCEF (SEQ ID NO: 117) (SEQ ID NO: 118) H2L8a QVQLVESGGTLVQPGGSLRLSCAASGFSFT QVQLVESGGTLVQPGGSLRLSCAASGFSFT DAWMSWVRQAPGKELEWVSSISGSGGSTYY DAWMSWVRQAPGKELEWVSSISGSGGSTYY AGSVKGRFTISRDNSKNTLYLQMNSLRAED AGSVKGRFTISRDNSKNTLYLQMNSLRAED TAVYYCARAAAAAAAAAAAAAAWGQGTLVT TAVYYCARAAAAAAAAAAAAAAWGQGTLVT VDDDDKEVQLVQSGGGLVQPGGSLRLSCAG VDDDDKEVQLVQSGGGLVQPGGSLRLSCAG SGFTFSSYVMHWLRQAPGKGLEWVSVIGTG SGFTFSSYVMHWLRQAPGKGLEWVSVIGTG GVTHYADSVKGRFTISRDNAKNSLYLQMNS GVTHYADSVKGRFTISRDNAKNSLYLQMNS LRAEDTAVYYCARWGYYGSGSYENDAFDIW LRAEDTAVYYCARWGYYGSGSYENDAFDIW GQGTMVTVSSASTKGPSVFPLAPSSKSTSG GQGTMVTVSSASTKGPSVFPLAPSSKSTSG GTAALGCLVKDYFPEPVTVSWNSGALTSGV GTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSVVTVPSSSLGTQT HTFPAVLQSSGLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKKVEPKCEF YICNVNHKPSNTKVDKKVEPKCEF (SEQ ID NO: 119) (SEQ ID NO: 120)

Example 3 TF-Binding ELISA

Biotinylated TF (1 μg/ml) was added to streptavidin pre-coated 96-well plates (Pierce Chemical, Rockford, Ill.) and incubated for 1 hr. The plates were then washed (5×) with PBS containing 0.5% Tween-20. Samples and controls (serially diluted) were added to the wells and incubated for 1 hr, followed by washes (5×) with PBS containing 0.5% Tween-20. Horseradish peroxidase (HRP)-conjugated anti-human IgG or HRP-conjugated anti-human Fab were diluted in PBS (1:5000) and added to each well. Following a 1 hr incubation, the plates were washed again. Amplex Red (10 μg/ml) was added to each well, and the signal was read using a plate reader. The data was analyzed using Softmax (Molecular Devices, Sunnyvale, Calif.). Results are shown in FIG. 10.

Example 4 RG1-Binding ELISA

Ninety-six well plates were coated with RG1 (1 μg/ml) by overnight incubation, and the plates were then washed (5×) with PBS containing 0.5% Tween-20. Samples and controls (serially diluted) were added to the wells and incubated for 1 hr, followed by washes (5×) with PBS containing 0.5% Tween-20. Horserandish peroxidase (HRP)-conjugated anti-human IgG or HRP-conjugated anti-human Fab were diluted in PBS (1:5000) and added to each well. Following a 1 hr incubation, the plates were washed again. Amplex Red (10 μg/ml) was added to each well, and the signal was read using a plate reader. The data was analyzed using Softmax (Molecular Devices, Sunnyvale, Calif.). Results are shown in FIG. 11.

Example 5 Sandwich Antigen-Binding ELISA

The antigen binding activity of a bispecific protease-regulated antibody (illustrated in FIG. 2) was measured using a sandwich antigen-binding ELISA. This antibody binds two antigens, RG-1 and TF, and the linker contains cleavage sites for enterokinase (“EK”).

Ninety-six well plates were coated with RG1 (1 μg/ml) by overnight incubation. The plates were then washed five times with PBS containing 0.5% Tween-20. Antibody samples and controls were digested with 30 units of enterokinase for 16 hr at 37° C. (see Example 4). The antibody samples, with or without enterokinase digestion, were serially diluted and added to the wells of the ELISA plates. The samples were incubated for one hour, followed by washes (5×) with PBS containing 0.5% Tween-20. Biotinylated TF (0.1 μg/ml) was added to each well and incubated for one hour. Horseradish peroxidase (HRP)-conjugated streptavidin (1:10000 diluted) was then added to each well. Following a one-hour incubation, the plates were washed again. Amplex Red (10 μg/ml) was added to each well, and the signal was read using a plate reader. The data was analyzed using Softmax® (Molecular Devices, Sunnyvale, Calif.). Parental antibodies 3E10, 19G9, and polyclonal human Fab were used as controls. The results are the average of duplicate wells (FIG. 12). The untreated bispecific protease-regulated antibody simultaneously binds to both TF and RG-1 (“Link 1” and “Link2,” respectively). However, following enterokinase treatment, the binding to both antigens is greatly reduced (“Link1/EK” and “Link2/EK,” respectively).

The antigen binding activity of several examples of protease-regulated antibodies was also measured using this assay. For example, the antigen binding activity of protease-regulated antibodies 3E10-Type1-Fab and 19G9-Type1-Fab is shown in FIG. 13. The controls are designated 3E10-Reg-Fab, 19G9-Reg-Fab, and HuFab.

The antigen binding activity of Fab-like protease-regulated antibodies is demonstrated in FIG. 14. The activity of antibodies H1L1, H1L4, H1L7, H4L7, and H5L5 (Type 2) was measured in the absence and presence of enterokinase. Parental antibodies 3E10, 19G9, and polyclonal human Fab were used as controls. Similarly, FIG. 15 shows the antigen binding activity of Fab-like protease-regulated antibodies H2L1, H2L2, and H2L8 (Type 3) and H3L1, H3L4, and H5L4 (Type 4).

Example 6 Enterokinase Digestion of Protease-Regulated Antibodies

Protease-regulated antibodies were digested with EnterokinaseMax™, the catalytic subunit of enterokinase (Invitrogen, Carlsbad, Calif.). The concentration of antibodies was adjusted to 1-5 μg/ml. A volume of antibody (100 μl) was mixed with 20 μl 10× EnterokinaseMax™ buffer and 75 μl sterile water in a tube. EnterokinaseMax™ (5 μl) was added to each sample and the samples were incubated at 37° C. for 16 hr. For the control group, a volume of water (5 μl) was used.

Example 7 Western Blots of Antibodies

Three detection antibodies were used to probe protease-regulated antibodies: anti-human kappa antibody, anti-human IgG(H+L), and anti-Myc tag antibodies. These detection antibodies were conjugated with horseradish peroxidase (HRP). Approximately 50 ng of antibody samples were mixed with loading buffer containing DTT (Invitrogen, Carlsbad, Calif.) and boiled for 5 min. The samples were then loaded onto a 12% Bis-Tris NuPAGE® gel (Invitrogen, Carlsbad, Calif.), separated, and transferred to nitrocellulose membranes. After blocking with 5% dry milk for 2 hr, the nitrocellulose membrane was incubated with a detection antibody for 1.5 hr. The membrane was then washed in PBS containing 0.5% Tween-20, and incubated with SuperSignal West Femto (Pierce Chemical, Rockford, Ill.), and expose to X-ray film for development. Results are shown in FIGS. 16-18.

Example 8 Subcutaneous Xenograft Cancer Model

Human mammary xenograft, MaTu cells are maintained as adherent cultures in RPMI supplemented with 10% FBS. Ncr nude mice (8-12 weeks of age) are inoculated subcutaneously in the right flank with 5×10⁶ cells in 0.1 mL of 80% matrigel/20% HBSS. When tumors reach an average size of ˜180 mg (6 days), treatment is initiated. Antibodies are administered i.v. once every four days (Q4Dx3) at a dose of 10 mg/kg. Control mice are treated with PBS or an unconjugated monoclonal antibody. Daily examinations into the health status of each animal are conducted. Each experimental group consists of 10 mice and the dosing volume was 0.1 mL/10 g body weight. The length and width of each tumor is measured by using an electronic caliper 2-3 times per week and tumor weights (mg) are calculated based on the formula of [length (mm)×width (mm)²]/2. All data, including daily observations, obtained throughout the course of the study are documented. Tumor growth inhibition (TGI) is calculated as 1−T/C×100, where T=final tumor weights from a treated group, and C=final tumor weights from the control group. The data demonstrates the therapeutic utility of antibodies for the treatment of tumors.

Other embodiments of the invention will be apparent to the skilled in the art from a consideration of this specification or practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with the true scope and spirit of the invention being indicated by the following claims. 

1. An antibody comprising one or more variable regions, wherein said antibody binds one or more antigens or epitopes.
 2. The antibody of claim 1, wherein said antibody comprises two variable regions, and wherein a first variable domain V1 is specific for a first antigen, a second variable domain V2 is specific for a second antigen
 3. The antibody of claim 2, wherein the first variable domain V1 comprises VL1 and VH1 domains which are specific for a first antigen, and wherein the second variable domain V2 comprises VL2 and VH2 domains which are specific for a second antigen.
 4. The antibody of claim 1, wherein the antibody is of the Fab-like or IgG-like type.
 5. The antibody of claim 1, wherein heavy and light chains are selected from the group consisting of: a) heavy chain: NH2-V_(L)1-V_(H)2-CH1-CH2-CH3-COOH light chain: NH2-V_(H)1-V_(L)2-CL-COOH, b) heavy chain: NH2-V_(H)1-V_(L)2-CH1-CH2-CH3-COOH light chain: NH2-V_(L)1-V_(H)2-CL-COOH, c) heavy chain: NH2-V_(L)1-V_(L)2-CH1-CH2-CH3-COOH light chain: NH2-V_(H)1-V_(H)2-CL-COOH, d) heavy chain: NH2-V_(L)1-V_(H)2-CH1-COOH light chain: NH2-V_(H)1-V_(L)2-CL-COOH, e) heavy chain: NH2-V_(H)1-V_(L)2-CH1-COOH light chain: NH2-V_(L)1-V_(H)2-CL-COOH, f) heavy chain: NH2-V_(L)1-V_(L)2-CH1-COOH light chain: NH2-V_(H)1-V_(H)2-CL-COOH, g) heavy chain: NH2-V_(H)1-V_(H)2-CH1-COOH light chain: NH2-V_(L)1-V_(L)2-CL-COOH, h) heavy chain: NH2-V_(H)1-V_(H)2-CH1-CH2-CH3-COOH, light chain: NH2-V_(L)1-V_(L)2-CL-COOH, i) heavy chain: NH2-V_(H)1-C_(H)1-CH2-CH3-COOH, light chain: NH2-V_(L)1-CL-COOH, and j) heavy chain: NH2-V_(L)1-C_(H)1-CH2-CH3-COOH, light chain: NH2-V_(H)1-CL-COOH.


6. The antibody of claim 1, further comprising a linker, wherein said linker comprises two or more amino acids.
 7. The linker of claim 6, wherein said linker comprises one or more protease cleavage sites.
 8. The linker of claim 7, wherein said protease cleavage sites are selected from the group consisting of SEQ ID NO: 1-32.
 9. The antibody of claim 6, wherein heavy and light chains are selected from the group consisting of: a) heavy chain: NH2-V_(L)1-linker-V_(H)2-CH1-CH2-CH3-COOH light chain: NH2-V_(H)1-linker-V_(L)2-CL-COOH, b) heavy chain: NH2-V_(H)1-linker-V_(L)2-CH1-CH2-CH3-COOH light chain: NH2-V_(L)1-linker-V_(H)2-CL-COOH, c) heavy chain: NH2-V_(L)1-linker-V_(L)2-CH1-CH2-CH3-COOH light chain: NH2-V_(H)1-linker-V_(H)2-CL-COOH, d) heavy chain: NH2-V_(L)1-linker-V_(H)2-C_(H)1-COOH light chain: NH2-V_(H)1-linker-V_(L)2-CL-COOH, e) heavy chain: NH2-V_(H)1-linker-V_(L)2-C_(H)1-COOH light chain: NH2-V_(L)1-linker-V_(H)2-CL-COOH, f) heavy chain: NH2-V_(L)1-linker-V_(L)2-C_(H)1-COOH light chain: NH2-V_(H)1-linker-V_(H)2-CL-COOH, g) heavy chain: NH2-V_(H)1-linker-V_(H)2-C_(H)1-COOH light chain: NH2-V_(L)1-linker-V_(L)2-CL-COOH, and h) heavy chain: NH2-V_(H)1-linker-V_(H)2-C_(H)1-CH2-CH3-COOH, light chain: NH2-V_(L)1-linker-V_(L)2-CL-COOH.


10. The antibody of claim 6, wherein the antibody simultaneously binds to two different antigens or two different epitopes.
 11. The antibody of claim 6, wherein the antibody sequentially binds to two antigens in a protease-dependent manner.
 12. The antibody of claim 6, wherein the antibody binds to antigen following protease digestion.
 13. The antibody of claim 1, selected from the group consisting of SEQ ID NO: 33-120.
 14. The antibody of claim 1, wherein said antibody is conjugated to a therapeutic or cytotoxic agent.
 15. The antibody of claim 14, wherein said antibody is conjugated to an agent selected from the group consisting of monomethylauristatin-E (MMAE), aplidin, azaribine, anastrozole, azacytidine, bleomycin, bortezomib, bryostatin-1, busulfan, calicheamycin, camptothecin, 10-hydroxycamptothecin, carmustine, celebrex, chlorambucil, cisplatin, irinotecan (CPT-I1), SN-38, carboplatin, cladribine, cyclophosphamide, cytarabine, dacarbazine, docetaxel, dactinomycin, daunomycin glucuronide, daunorubicin, dexamethasone, diethylstilbestrol, doxorubicin, doxorubicin glucuronide, epirubicin glucuronide, ethinyl estradiol, estramustine, etoposide, etoposide glucuronide, etoposide phosphate, floxuridine (FUdR), 3′,5′-O-dioleoyl-FudR (FUdR-dO), fludarabine, flutamide, fluorouracil, fluoxymesterone, gemcitabine, hydroxyprogesterone caproate, hydroxyurea, idarubicin, ifosfamide, L-asparaginase, leucovorin, lomustine, mechlorethamine, medroprogesterone acetate, megestrol acetate, melphalan, mercaptopurine, 6-mercaptopurine, methotrexate, mitoxantrone, mithramycin, mitomycin, mitotane, phenyl butyrate, prednisone, procarbazine, paclitaxel, pentostatin, PSI-341, semustine streptozocin, tamoxifen, taxanes, taxol, testosterone propionate, thalidomide, thioguanine, thiotepa, teniposide, topotecan, uracil mustard, velcade, vinblastine, vinorelbine, vincristine, ricin, abrin, ribomiclease, onconase, rapLR1, DNase I, Staphylococcal enterotoxin-A, pokeweed antiviral protein, gelonin, diphtheria toxin, Pseudomonas exotoxin, and Pseudomonas endotoxin, and functional analogs thereof
 16. A pharmaceutical composition comprising a therapeutically effective amount of an antibody of claim 1, in combination with a pharmaceutically acceptable carrier.
 17. The pharmaceutical composition of claim 16 further comprising one or more pharmaceutical agents.
 18. A method for the treatment of cancer, infectious diseases, and autoimmune diseases comprising administering to a subject in need an effective amount of an antibody of claim
 1. 19. The method of claim 18, wherein the cancer is selected from cancers of the breast, respiratory tract, brain, reproductive organs, digestive tract, urinary tract, eye, liver, skin, head and neck, thyroid, parathyroid, lymphomas, sarcomas, and leukemias.
 20. The method of claim 18, wherein the autoimmune disease is selected from multiple sclerosis, rheumatoid arthritis, lupus, type I diabetes mellitus, Crohn's disease, autoimmune hemolytic anemia, autoimmune hepatitis, glomerulonephritis, inflammatory bowel disease, myocarditis, psoriasis, thyroiditis, ulcerative colitis, and Graves'disease.
 21. The method of claim 18, wherein the infectious disease is selected from HIV/AIDS, lower respiratory infections, diarrheal diseases, tuberculosis, malaria, measles, pertussis, tetanus, meningitis, syphilis, hepatitis B and tropical diseases. 22-24. (canceled)
 25. A diagnostic method to detect a disease in a patient comprising: (a) immunologically detecting and quantifying the level of a protein associated with a disease in control samples taken from individuals of a control population; (b) immunologically detecting and quantifying changes in the protein in samples of a patient sample taken from a patient over time; and (c) comparing the levels of the protein in the patient's samples to the level of the protein in the control samples; wherein a level of the protein in the patient's samples that is above the level of the protein in the control samples is indicative the presence of disease in the patient.
 26. A method for monitoring the status of a disease in a patient, and/or monitoring how a patient with said disease is responding to a therapy comprising immunologically detecting and quantifying changes in the levels of a protein associated with a disease in patient samples taken over time, wherein increasing levels of the protein over time indicate disease progression or a negative response to said therapy, and wherein decreasing levels of the protein over time indicate disease remission or a positive response to said therapy.
 27. A kit comprising an antibody of claim
 1. 28. The kit of claim 27, further comprising solutions for suspending or fixing the cells, detectable labels, solutions for rendering a polypeptide susceptible to the binding of an antibody, solutions for lysing cells, and/or solutions for the purification of polypeptides. 